WO2023190560A1 - Method, composition, and kit for detecting human b-type natriuretic peptide, or precursor or degradation product of same - Google Patents

Abstract

A method for detecting hBNP, or a precursor or a degradation product of the same, in a sample according to one aspect of the present invention comprises a step for detecting, by a latex coagulating method, hBNP, or a precursor or a degradation product of the same, in the sample by using: a monoclonal antibody that recognizes an amino acid region at positions 17-24 in the amino acid sequence indicated by SEQ ID NO. 1, or an antigen binding fragment of the monoclonal antibody; and a monoclonal antibody that recognizes an amino acid region at positons 5-13 in the amino acid sequence indicated by SEQ ID NO. 1, or an antigen binding fragment of the monoclonal antibody. The precursor of hBNP is hproBNP, and the degradation product of hBNP is a degradation product including an amino acid region at positions 5-24 in the amino acid sequence indicated by SEQ ID NO. 1. According to the present invention, it is possible to detect hBNP by using a latex coagulating method while suppressing effects caused by steric hindrance of antibody-immobilized latex particles and degradation of terminals of hBNP.

Description

Methods, compositions, and kits for detecting human B-type natriuretic peptide or precursors or degradation products thereof

The present invention relates to methods, compositions, and kits for detecting human B-type natriuretic peptide or its precursors or degradation products.

Human B-type natriuretic peptide (hBNP, SEQ ID NO: 1) is a circulating hormone that is biosynthesized and secreted by cardiac muscle cells, and has effects such as diuresis, vasodilation, sympathoinhibition, and cardiac hypertrophy suppression. , works to protect the heart muscle. When the heart (particularly the ventricle) is under stress, human preproB-type natriuretic peptide (hpreproBNP, SEQ ID NO: 2), which is one of the precursors of hBNP, is first expressed, and human proB-type natriuretic peptide (hproBNP, SEQ ID NO: 3) is expressed. ) and undergo post-translational modification. After that, the peptide bond between the 96th amino acid (arginine) and the 97th amino acid (serine) of hproBNP is enzymatically cleaved, producing hBNP consisting of amino acids 97 to 128 (32 amino acids). and fulfills the above-mentioned pathophysiological functions. Therefore, the expression level of hBNP increases depending on the increased workload of the heart and the disease state, and is reflected in the blood concentration. As described above, hBNP expression is enhanced depending on the severity of heart failure, and blood hBNP increases, so hBNP has been used as a diagnostic marker for heart failure.

hBNP is said to have a biological half-life of about 20 minutes in the body, and is known to be an unstable molecule in plasma and serum. Degradation of hBNP by protease-type enzymes has been reported, and for example, in Non-Patent Document 1, it is reported that the Arg30-Arg31 bond at the C-terminus and the N-terminal portion, more specifically, the Pro2-Lys3 bond, is cleaved. Are listed. It has been reported that the resulting hBNP degradation product retains the above-mentioned pathophysiological function, and considering the mechanism of expression of compensatory responses to the disease state of heart failure, total hBNP, including hBNP degradation product, is Evaluating the intermediate concentration is important as a diagnostic index that contributes to understanding the pathology of heart failure and subsequent treatment (Non-Patent Document 2).

A sandwich immunoassay method using two or more monoclonal or polyclonal antibodies that recognize different epitopes of hBNP has conventionally been used to detect hBNP. Sandwich immunoassay is a method generally used for quantitative and qualitative detection of antigens. In such methods, antibodies that recognize each epitope are simultaneously bound to different epitopes on the antigen, or one antibody is bound and then the other antibody is bound, and the antigen sandwiched by the two antibodies is bound. To detect.

As anti-hBNP antibodies that can be used in conventional sandwich immunoassay methods, antibodies that bind to various epitopes have been reported. For example, Patent Document 1 describes a monoclonal antibody that recognizes histidine (His32), which is the last amino acid of the C-terminal epitope (Lys27 to His32) of hBNP. As other epitopes on hBNP, Patent Document 2 describes Ser1 to Cys10, Val5 to Arg13, and Met15 to Gly25.

Patent No. 2665850 Japanese Patent Application Publication No. 2007-169293

Shimizu, et al. (2002) Clinica Chimica Acta 316:129-135 Tomoko Ichiki, et al. Adv Clin Chem. 2013;61:1-31

Since high sensitivity is required for the detection of hBNP, a method using chemiluminescence (chemiluminescent enzyme immunoassay) has been used among sandwich immunoassay methods. Since chemiluminescent reagents are expensive, it would be preferable to utilize a cheaper latex agglutination method. However, in the latex agglutination method, it is necessary to immobilize two types of antibodies on latex particles, and since hBNP is a small peptide consisting of 32 amino acids, when conventional anti-hBNP antibodies are used, the antibody Antibodies may not be able to bind to antigens due to steric hindrance between immobilized latex particles. In order to avoid steric hindrance between antibody-immobilized latex particles, it is possible to use an antibody that recognizes the C-terminus of hBNP as one of the two types of antibodies, but in this case, the terminal Decomposed hBNP cannot be detected.

The present invention was made in view of the above problems, and aims to detect hBNP by a latex agglutination method while suppressing the effects of steric hindrance between antibody-immobilized latex particles and decomposition of the terminals of hBNP.

As a result of extensive studies, the present inventors discovered a novel epitope hBNP17-24 (17th to 24th amino acid region of hBNP: SEQ ID NO: 4) in a region other than the N-terminal and C-terminal regions of hBNP that are susceptible to protease degradation. We then created a new antibody that recognizes this epitope. Surprisingly, although hBNP17-24 is located near the known epitope hBNP5-13 (5th to 13th amino acid region of hBNP: SEQ ID NO: 5), the antibody that recognizes hBNP17-24 and hBNP5 By using a combination of antibodies that recognize -13, it was possible to efficiently detect hBNP by latex agglutination method without being affected by steric hindrance. The combination of the above antibodies is hardly affected by the degradation of the N-terminus and C-terminus of hBNP. The present inventors have completed the present invention based on the above findings.

A method for detecting human B-type natriuretic peptide or its precursor or decomposition product in a sample according to one aspect of the present invention uses a monoclonal antibody that recognizes the 17th to 24th amino acid region in the amino acid sequence shown by SEQ ID NO: 1. or human B-type natriuretic peptide in a sample using an antigen-binding fragment thereof and a monoclonal antibody or antigen-binding fragment thereof that recognizes the 5th to 13th amino acid region in the amino acid sequence shown by SEQ ID NO: 1. or a step of detecting its precursor or decomposition product by a latex agglutination method, the precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and the decomposition product of human B-type natriuretic peptide is SEQ ID NO: 1. This is a decomposition product containing the 5th to 24th amino acid region in the amino acid sequence shown.

A composition for detecting human B-type natriuretic peptide or its precursor or decomposition product according to one aspect of the present invention is a monoclonal antibody that recognizes the 17th to 24th amino acid region in the amino acid sequence shown by SEQ ID NO: 1. or a first latex particle on which an antigen-binding fragment thereof is immobilized, and a second latex particle on which a monoclonal antibody that recognizes the 5th to 13th amino acid region in the amino acid sequence shown by SEQ ID NO: 1 or an antigen-binding fragment thereof is immobilized. The precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and the decomposition product of human B-type natriuretic peptide is the 5th to 24th latex particles in the amino acid sequence shown in SEQ ID NO: 1. It is a decomposition product containing the amino acid region of

A kit for detecting human B-type natriuretic peptide or its precursor or decomposition product according to one aspect of the present invention includes a monoclonal antibody or A first latex particle on which the antigen-binding fragment is immobilized, and a second latex particle on which a monoclonal antibody that recognizes the 5th to 13th amino acid region in the amino acid sequence shown by SEQ ID NO: 1 or an antigen-binding fragment thereof is immobilized. latex particles, the precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and the decomposition product of human B-type natriuretic peptide consists of the 5th to 24th amino acids in the amino acid sequence shown in SEQ ID NO: 1. It is a decomposition product containing an amino acid region.

According to the present invention, hBNP can be detected by a latex agglutination method while suppressing the effects of steric hindrance between antibody-immobilized latex particles and decomposition of the terminals of hBNP.

FIG. 2 is a diagram showing the results of epitope mapping of antibodies that specifically bound to hBNP. FIG. 3 shows the reactivity of different antibody pairs immobilized on latex particles with hBNP. Figure 2 (A) shows the results using an antibody pair that recognizes the 5th to 13th and 17th to 24th amino acid regions of hBNP, and Figure 2 (B) shows the results using the antibody pair that recognizes the 5th to 13th and 17th to 24th amino acid regions of hBNP. These are the results using an antibody pair that recognizes the ~20th amino acid region. FIG. 3 is a diagram showing the influence of hBNP degradation in plasma on the reactivity of different antibody pairs immobilized on latex particles with hBNP. FIG. 3 (A) shows the results using an antibody pair that recognizes the 5th to 13th and 17th to 24th amino acid regions of hBNP, and FIG. 3 (B) shows the results using the antibody pair that recognizes the 5th to 13th and 17th to These are the results using an antibody pair that recognizes the ~32nd amino acid region.

<Detection method>
A method for detecting human B-type natriuretic peptide (hBNP) or its precursor or decomposition product in a sample according to one aspect of the present invention includes the 17th to 24th amino acid region (sequence No. 4), a monoclonal antibody or antigen-binding fragment thereof that recognizes the 5th to 13th amino acid region (SEQ ID NO: 5) in the amino acid sequence shown by SEQ ID NO: 1, The method includes the step of detecting human B-type natriuretic peptide or its precursor or decomposition product in a sample by a latex agglutination method using a latex agglutination method.

The amino acid sequence shown in SEQ ID NO: 1 is the full-length amino acid sequence of hBNP, and a monoclonal antibody that recognizes the 17th to 24th amino acid region in the amino acid sequence shown in SEQ ID NO: 1 and the 5th to 24th amino acids in the amino acid sequence shown in SEQ ID NO: 1 Monoclonal antibodies that recognize the 13th amino acid region are hBNP17-24 epitope (17th to 24th amino acid region of hBNP: SEQ ID NO: 4) and hBNP5-13 epitope (5th to 13th amino acid region of hBNP: This is a monoclonal antibody that specifically binds to SEQ ID NO: 5). Here, "specifically" means that in a liquid system in which the protein having the hBNP epitope, other protein components not having the epitope, and the monoclonal antibody are mixed, the monoclonal antibody Even if no antigen-antibody reaction occurs with the component at a detectable level, or even if some binding reaction or association reaction occurs, the reaction is clearly weaker than the antigen-antibody reaction of the monoclonal antibody with the protein having the above epitope. It means not to wake up.

A monoclonal antibody that recognizes the 17th to 24th amino acid region of hBNP and a monoclonal antibody that recognizes the 5th to 13th amino acid region of hBNP can also bind to the precursor or degraded product of hBNP. The precursor of hBNP is human pro-B-type natriuretic peptide (hproBNP). The degradation product of hBNP is not particularly limited as long as it includes the 5th to 24th amino acid region in the amino acid sequence of hBNP. The degradation product of hBNP may be, for example, a degradation product in which the 31st and 32nd amino acids are removed from hBNP, or a degradation product in which the 1st and 2nd amino acids are removed from hBNP.

The class of the monoclonal antibody is not limited to IgG, but may be IgY, IgM, camel Ig, or Ig NAR. Further, the antigen-binding fragment of the monoclonal antibody is not particularly limited, and may be Fab, Fab', F(ab') ₂ , or single chain antibody (scFv).

The above monoclonal antibody can be obtained, for example, by immunizing an animal with hBNP or a hBNP fragment containing the above epitope using a known immunological technique, and from a hybridoma produced using cells of the immunized animal. Alternatively, the monoclonal antibodies described above can also be produced by genetic recombination techniques such as phage display methods and yeast display methods. The length of the hBNP fragment used for immunization is not particularly limited, but is preferably 10 amino acids or more, more preferably 13 amino acids or more.

The sample is not limited as long as it may contain hBNP or its precursor or decomposed product, and may be a biological sample collected from a human or a non-human animal, for example. The biological sample may be, for example, blood (whole blood), plasma, or serum.

Using a monoclonal antibody or antigen-binding fragment thereof that recognizes the 17th to 24th amino acid region of hBNP and a monoclonal antibody or antigen-binding fragment thereof that recognizes the 5th to 13th amino acid region of hBNP, The step of detecting the human B-type natriuretic peptide or its precursor or decomposition product by a latex agglutination method can be performed according to a known latex agglutination method. That is, first, a monoclonal antibody or antigen-binding fragment thereof that recognizes the 17th to 24th amino acid region of hBNP and a monoclonal antibody or antigen-binding fragment thereof that recognizes the 5th to 13th amino acid region of hBNP are respectively It is immobilized on one latex particle and a second latex particle. Next, if necessary, each antibody-immobilized latex particle is blocked using a known blocking agent such as bovine serum albumin (BSA), and then the antibody-immobilized latex particle and sample are mixed in a buffer solution to form a mixed solution. are incubated at a temperature that allows antigen-antibody reactions to occur. As a result, if hBNP or its precursor or decomposed product is present in the sample, antibody-immobilized latex particles will aggregate due to an antigen-antibody reaction, so the presence or absence of this aggregation can be determined by known methods such as light irradiation. By detecting this, it is possible to detect the presence or absence of hBNP or its precursor or decomposition product in the sample.

The first latex particles and the second latex particles may be the same or different, and known latex particles can be used. The materials of the first latex particles and the second latex particles are not particularly limited, and may be, for example, polystyrene, polyacrylonitrile, polymethacrylonitrile, or polymethyl methacrylate. The average particle diameter of the first latex particles and the second latex particles is, for example, 0.02 to 5 μm, 0.05 to 1 μm, or 0.2 μm, from the viewpoint of reducing steric hindrance and obtaining sufficient detection sensitivity. ~0.6 μm. In this specification, the average particle size refers to the particle size ( In other words, it means the median diameter).

The first latex particle has immobilized a monoclonal antibody that recognizes the 17th to 24th amino acid region of hBNP, and the second latex particle has immobilized a monoclonal antibody that recognizes the 5th to 13th amino acid region of hBNP. It may be mixed with the sample at the same time or separately with the sample. When these antibody-immobilized latex particles are mixed with a sample separately, one of the antibody-immobilized latex particles and the sample are mixed, the mixed solution is incubated at a temperature where an antigen-antibody reaction can occur, and then the other antibody-immobilized latex particles are mixed with the sample. of the antibody-immobilized latex particles can be mixed and re-incubated at a temperature at which antigen-antibody reactions can occur.

The temperature of incubation is not particularly limited, and may be, for example, 20 to 37°C, particularly 37°C, 25°C, or 20°C.

A monoclonal antibody that recognizes the 17th to 24th amino acid region of hBNP and a monoclonal antibody that recognizes the 5th to 13th amino acid region of hBNP are different from each other even though their epitopes are close to each other. It is possible to bind to hBNP with almost no influence of steric hindrance between immobilized latex particles. Therefore, according to the method according to this aspect, hBNP or its precursor or decomposition product can be detected by the latex aggregation method while suppressing the influence of steric hindrance. Furthermore, since the monoclonal antibody that recognizes the 17th to 24th amino acid region of hBNP and the monoclonal antibody that recognizes the 5th to 13th amino acid region of hBNP recognize a region other than the terminal region of hBNP, this aspect According to the method, hBNP or its precursor or decomposition product can be detected by the latex aggregation method while suppressing the influence of decomposition of the terminal of hBNP.

<Detection composition>
The composition for detecting hBNP or its precursor or decomposition product according to one aspect of the present invention is used in the detection method according to the above-mentioned aspect of the present invention. A first latex particle on which a monoclonal antibody that recognizes the amino acid region of SEQ ID NO. and second latex particles having immobilized sexual fragments. Details of these monoclonal antibodies and the first and second latex particles are as described above. The composition according to this aspect may be a buffer solution containing the antibody-immobilized latex particles. By mixing the composition according to this aspect with a sample, hBNP or its precursor or decomposition product in the sample can be detected by a latex agglutination method.

<Detection kit>
The composition according to the above aspect comprises a first latex particle on which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the 17th to 24th amino acid region in the amino acid sequence shown in SEQ ID NO: 1 is immobilized; In another aspect of the present invention, a composition comprising second latex particles on which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the 5th to 13th amino acid region in the shown amino acid sequence is immobilized, These antibody-immobilized latex particles may be provided as separate reagents. That is, another aspect of the present invention is to provide a first latex particle on which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the 17th to 24th amino acid region in the amino acid sequence shown by SEQ ID NO: 1 is immobilized; A second latex particle on which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the 5th to 13th amino acid region in the amino acid sequence shown by number 1 is immobilized, and detects hBNP or its precursor or decomposition product. This is a kit for

The kit can further include known reagents, materials, instruments, etc. used in latex agglutination methods, such as buffers.

<Test Example 1> Epitope mapping of novel antibodies Immunization of mice and rabbits Peptides 1 to 19 shown in Table 1 were synthesized, and each peptide was modified with various functional groups to increase immunogenicity. It was bound to carrier proteins such as globulin, KLH (keyhole limpet hemocyanin), OVA (ovalbumin), and BSA. Mice and rabbits were prepared for each peptide conjugated to a carrier protein and immunized for 2 months at intervals of 7 to 14 days. As an adjuvant, sigma adjuvant system (registered trademark) (manufactured by Merck & Co., Ltd.) was used. Blood was collected over time during the immunization period, and the antibody titer was measured by ELISA (enzyme-linked immunosorbent assay). Individuals with high antibody titers were selected and their spleens were removed.

Antibody acquisition RNA was extracted from the removed spleen using TRIzol (registered trademark) Plus RNA Purification Kit (manufactured by Thermo Fisher Scientific) and SuperScript (registered trademark) III Reverse Transcriptase (manufactured by Thermo Fisher Scientific). The cDNA was obtained by the reverse transcription reaction used. The obtained cDNA was amplified by PCR reaction using antibody gene-specific primers, and the amplified product was inserted into a vector to obtain an antibody gene library. Using the library and hBNP-immobilized beads, four rounds of panning were performed using the phage display method, and antibodies that specifically bind to hBNP were confirmed using the ELISA method. The sequences of various antibodies that specifically bind to hBNP were inserted into cell expression vectors, expressed in CHO (Chinese Hamster Ovary) cells, and then purified using a Protein A (GE) column. After obtaining full-length antibodies, epitopes were identified for all of them. Note that most of the 18 types of antibodies obtained were derived from individuals immunized with peptide 19.

Epitope Identification The epitope of the obtained antibody was confirmed as follows. First, biotinylated peptides 1 to 18 were synthesized (outsourced to JPT Peptide Technologies) and immobilized on mutually distinguishable beads (LumAvidin® Microspheres manufactured by Luminex). 500 of each peptide-immobilized beads were added to each well of a polypropylene microplate, and one type of antibody was added to each well at a final concentration of 1 μg/mL. After incubating the microplate at 37°C for 1 hour, the supernatant was removed using a magnetic stand and the microplate was washed. R-phycoerythrin-labeled goat anti-mouse IgG or goat anti-rabbit IgG was added as a detection antibody and incubated at 37°C for 1 hour. After removing the supernatant using a magnetic stand and washing the microplate, the fluorescence of R-phycoerythrin was measured using Bio-Plex. The results of epitope mapping of one of the antibodies derived from mice immunized with peptide 19 are shown in FIG.

A significantly larger number of antibodies bound to peptides containing the 17th and 24th amino acids of hBNP (peptides 10 to 17) than to peptides that do not contain these amino acids. This result suggested that this antibody specifically recognizes the 17th to 24th amino acid region of hBNP.

<Test Example 2> Evaluation of the influence of steric hindrance Various anti-hBNP antibody solutions were mixed with polystyrene beads (average particle size: 0.3 μm) and reacted overnight at 4° C. to obtain antibody-immobilized beads. As antibodies, an antibody that recognizes the 5th to 13th amino acid region of hBNP, an antibody that recognizes the 17th to 24th amino acid region of hBNP, and an antibody that recognizes the 14th to 20th amino acid region of hBNP were used. After further adding BSA to each antibody-immobilized bead and performing blocking, beads on which an antibody that recognizes the 5th to 13th amino acid region of hBNP was immobilized and the 17th to 24th or 14th to 20th amino acid regions of hBNP were added. Beads with immobilized antibodies that recognize the region were suspended in a buffer solution. Synthetic hBNP (manufactured by Prospec) diluted to 10 ng/mL with PBS was added to the dilution buffer and allowed to stand at 37°C for 5 minutes, then mixed with the antibody-immobilized bead suspension and reacted at 37°C for 5 minutes. The latex agglutination reaction was detected by irradiation with 635 nm light. The results are shown in Figure 2.

Figure 2 (A) shows the results using an antibody pair that recognizes the 5th to 13th and 17th to 24th amino acid regions of hBNP, and Figure 2 (B) shows the results using the antibody pair that recognizes the 5th to 13th and 17th to 24th amino acid regions of hBNP. These are the results using an antibody pair that recognizes the ~20th amino acid region. When antibody pairs recognizing the 5th to 13th and 17th to 24th amino acid regions were used, the scattering intensity of 635 nm light increased over time; When using an antibody pair that recognizes the amino acid region of , the scattering intensity increased only slightly. This result shows that when antibody pairs recognizing the 5th to 13th and 17th to 24th amino acid regions were used, no steric hindrance occurred between the antibody-immobilized latex particles and the latex reaction proceeded. When antibody pairs recognizing the 5th to 13th and 14th to 20th amino acid regions were used, the latex agglutination reaction was inhibited due to steric hindrance. The affinities for hBNP of the antibody that recognizes the 14th to 20th amino acid region and the antibody that recognizes the 17th to 24th amino acid region are 2.4 x 10 ^-10 M and 2.41 x 10 ^-10 M, respectively. This has been confirmed using Biacore (registered trademark) 8K (manufactured by Cytiva), and it is considered that the difference in aggregation is not due to a difference in affinity.

<Test Example 3> Evaluation of the influence of hBNP decomposition Various anti-hBNP antibody solutions were mixed with polystyrene beads (average particle size: 0.3 μm) and reacted overnight at 4°C to obtain antibody-immobilized beads. . As antibodies, an antibody that recognizes the 5th to 13th amino acid region of hBNP, an antibody that recognizes the 17th to 24th amino acid region of hBNP, and an antibody that recognizes the 27th to 32nd amino acid region of hBNP were used. After further adding BSA to each antibody-immobilized bead and performing blocking, beads on which an antibody that recognizes the 5th to 13th amino acid region of hBNP was immobilized and the 17th to 24th or 27th to 32nd amino acids of hBNP were added. Beads with immobilized antibodies that recognize the region were suspended in a buffer solution. Synthetic hBNP (manufactured by Prospec) diluted with hBNP-depleted plasma (manufactured by hytest) was added to the dilution buffer and reacted at 37°C for 5 minutes, then mixed with the antibody-immobilized bead suspension and incubated at 37°C. The reaction was allowed to proceed for 5 minutes, and the latex agglutination reaction was detected by irradiation with 730 nm light (first day). Furthermore, on the first day, synthetic hBNP diluted with hBNP-depleted plasma (manufactured by hytest) was allowed to stand overnight at 4°C, and this synthetic hBNP was added to the dilution buffer to perform a latex agglutination reaction as described above. (2nd day). The results are shown in Figure 3.

In FIG. 3, the scattering intensity is shown as the difference between the scattering intensity at 0 minutes after the start of the reaction and the scattering intensity at 5 minutes after the start of the reaction. FIG. 3 (A) shows the results using an antibody pair that recognizes the 5th to 13th and 17th to 24th amino acid regions of hBNP, and FIG. 3 (B) shows the results using the antibody pair that recognizes the 5th to 13th and 17th to These are the results using an antibody pair that recognizes the ~32nd amino acid region. When an antibody pair recognizing amino acid regions 5 to 13 and 17 to 24 was used, latex agglutination reactions occurred on both the 1st and 2nd day, whereas When an antibody pair recognizing the amino acid region of was used, a latex agglutination reaction occurred only on the first day, and no latex agglutination reaction occurred on the second day. This result indicates that hBNP stored in plasma overnight has its terminals degraded, and the antibody pair that recognizes the 5th to 13th and 17th to 24th amino acid regions detects hBNP with degraded terminals. In contrast, the antibody pairs that recognize amino acid regions 5 to 13 and 27 to 32 cannot detect hBNP with degraded terminals.

Claims (3)

A method for detecting human B-type natriuretic peptide or its precursor or decomposition product in a sample, the method comprising:
A monoclonal antibody or antigen-binding fragment thereof that recognizes the 17th to 24th amino acid region in the amino acid sequence shown by SEQ ID NO: 1,
A monoclonal antibody or antigen-binding fragment thereof that recognizes the 5th to 13th amino acid region in the amino acid sequence shown by SEQ ID NO: 1,
Detecting human B-type natriuretic peptide or its precursor or decomposition product in the sample by a latex agglutination method using
The precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and the decomposition product of human B-type natriuretic peptide is a decomposition product containing the 5th to 24th amino acid region in the amino acid sequence shown by SEQ ID NO: 1. There is a method. A composition for detecting human B-type natriuretic peptide or its precursor or degradation product, comprising:
A first latex particle on which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the 17th to 24th amino acid region in the amino acid sequence shown by SEQ ID NO: 1 is immobilized;
a second latex particle on which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the 5th to 13th amino acid region in the amino acid sequence shown by SEQ ID NO: 1 is immobilized;
The precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and the decomposition product of human B-type natriuretic peptide is a decomposition product containing the 5th to 24th amino acid region in the amino acid sequence shown by SEQ ID NO: 1. There is a composition. A kit for detecting human B-type natriuretic peptide or its precursor or decomposition product, comprising:
A first latex particle on which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the 17th to 24th amino acid region in the amino acid sequence shown by SEQ ID NO: 1 is immobilized;
a second latex particle on which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the 5th to 13th amino acid region in the amino acid sequence shown by SEQ ID NO: 1 is immobilized;
The precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and the decomposition product of human B-type natriuretic peptide is a decomposition product containing the 5th to 24th amino acid region in the amino acid sequence shown by SEQ ID NO: 1. Yes, there is a kit.

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