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

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

[Technical Field]

[0001] The present invention relates to methods, compositions, and kits for detecting human B-type natriuretic peptide or its precursors or degradation products. [Background technology]

[0002] Human B-type natriuretic peptide (hBNP, SEQ ID NO: 1) is a circulating hormone biosynthesized and secreted by cardiac myocytes. It has diuretic, vasodilatory, sympathostatic, and cardiac hypertrophy-inhibiting effects, thereby protecting the myocardium. When the heart (particularly the ventricle) is stressed, human prepro B-type natriuretic peptide (hpreproBNP, SEQ ID NO: 2), a precursor of hBNP, is first expressed and cleaved to human pro B-type natriuretic peptide (hproBNP, SEQ ID NO: 3), which undergoes post-translational modification. Subsequently, the peptide bond between the 96th amino acid (arginine) and the 97th amino acid (serine) of hproBNP is enzymatically cleaved to generate hBNP, which consists of amino acids 97-128 (32 amino acids), and exerts the pathophysiological functions described above. Therefore, the expression level of hBNP increases in response to increased cardiac stress and disease progression, which is reflected in its blood concentration. Thus, hBNP expression increases depending on the severity of heart failure, leading to an increase in blood hBNP levels, and hBNP has been used as a diagnostic marker for heart failure.

[0003] 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; for example, Non-Patent Document 1 describes the cleavage of the Arg30-Arg31 bond at the C-terminus and the N-terminus, more specifically the Pro2-Lys3 bond. It has been reported that the resulting hBNP degradation products also retain the above-mentioned pathophysiological functions. Considering the mechanism of expression of compensatory responses to the disease progression of heart failure, evaluating the blood concentration of total hBNP, including hBNP degradation products, is important as a diagnostic indicator that contributes to understanding the pathology of heart failure and subsequent treatment (Non-Patent Document 2).

[0004] For the detection of hBNP, sandwich immunoassays using two or more monoclonal or polyclonal antibodies that recognize different epitopes of hBNP have traditionally been used. Sandwich immunoassays are generally used for quantitative and qualitative detection of antigens. In such methods, antibodies that recognize different epitopes on the antigen are simultaneously bound to the antigen, or one antibody is bound to the antigen first, followed by the other antibody, and the antigen sandwiched between the two antibodies is detected.

[0005] Antibodies that bind to various epitopes have been reported as anti-hBNP antibodies that can be used in conventional sandwich immunoassays. For example, Patent Document 1 describes a monoclonal antibody that recognizes histidine (His32), the last amino acid of the C-terminal epitope (Lys27 to His32) of hBNP. Patent Document 2 describes other epitopes on hBNP, including Ser1 to Cys10, Val5 to Arg13, and Met15 to Gly25. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 2665850 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-169293 [Non-patent literature]

[0007] [Non-Patent Document 1] Shimizu, et al. (2002) Clinica Chimica Acta 316:129-135 [Non-patent document 2] Tomoko Ichiki, et al. Adv Clin Chem. 2013;61:1-31 Summary of the Invention [Problem to be solved by the invention]

[0008] Because high sensitivity is required for the detection of hBNP, sandwich immunoassays using chemiluminescence (chemiluminescent enzyme immunoassay) have been used. Because chemiluminescent reagents are expensive, the use of the less expensive latex agglutination method would be preferable. However, the latex agglutination method requires immobilization of two types of antibodies to latex particles. Furthermore, because hBNP is a small peptide consisting of 32 amino acids, steric hindrance between the antibody-immobilized latex particles can prevent the antibody from binding to the antigen when using conventional anti-hBNP antibodies. To avoid steric hindrance between the antibody-immobilized latex particles, it is possible to use an antibody that recognizes the C-terminus of hBNP as one of the two antibodies. However, in this case, hBNP whose terminal has been cleaved cannot be detected in serum or plasma.

[0009] The present invention has been made in consideration of the above-mentioned problems, and aims to detect hBNP by the latex agglutination method while suppressing the effects of steric hindrance between antibody-immobilized latex particles and terminal degradation of hBNP. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors discovered a novel epitope, hBNP17-24 (the 17th to 24th amino acid region of hBNP; SEQ ID NO: 4), in a region other than the N- and C-terminal regions of hBNP, which are susceptible to degradation by proteases, and produced a novel antibody that recognizes this epitope. Surprisingly, although hBNP17-24 is located near the known epitope, hBNP5-13 (the 5th to 13th amino acid region of hBNP; SEQ ID NO: 5), by using a combination of an antibody that recognizes hBNP17-24 and an antibody that recognizes hBNP5-13, hBNP could be efficiently detected by latex agglutination with almost no steric hindrance. This antibody combination is also almost unaffected by degradation of the N- and C-termini of hBNP. Based on these findings, the present inventors have completed the present invention.

[0011] A method for detecting human B-type natriuretic peptide or its precursor or cleavage products in a sample according to one aspect of the present invention comprises the step of detecting human B-type natriuretic peptide or its precursor or cleavage products in the sample by latex agglutination using 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, and a monoclonal antibody or an antigen-binding fragment thereof that recognizes the 5th to 13th amino acid region in the amino acid sequence shown in SEQ ID NO: 1, wherein the precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and the cleavage products of human B-type natriuretic peptide are cleavage products comprising the 5th to 24th amino acid region in the amino acid sequence shown in SEQ ID NO: 1.

[0012] A composition for detecting human B-type natriuretic peptide or its precursor or cleavage products according to one aspect of the present invention comprises first latex particles onto 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, and second latex particles onto 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 in SEQ ID NO: 1 is immobilized, wherein the precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and the cleavage product of human B-type natriuretic peptide is a cleavage product comprising the 5th to 24th amino acid region in the amino acid sequence shown in SEQ ID NO: 1.

[0013] A kit for detecting human B-type natriuretic peptide or its precursor or cleavage products according to one aspect of the present invention comprises first latex particles onto 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, and second latex particles onto 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 in SEQ ID NO: 1 is immobilized, wherein the precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and the cleavage product of human B-type natriuretic peptide is a cleavage product comprising the 5th to 24th amino acid region in the amino acid sequence shown in SEQ ID NO: 1. [Effects of the Invention]

[0014] According to the present invention, hBNP can be detected by the latex agglutination method while suppressing the effects of steric hindrance between antibody-immobilized latex particles and terminal degradation of hBNP. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the results of epitope mapping of antibodies that specifically bind to hBNP. [Figure 2]2 shows the reactivity of different antibody pairs immobilized on latex particles with hBNP. Figure 2(A) shows the results using antibody pairs that recognize the amino acid regions of positions 5 to 13 and positions 17 to 24 of hBNP, and Figure 2(B) shows the results using antibody pairs that recognize the amino acid regions of positions 5 to 13 and positions 14 to 20 of hBNP. [Figure 3] 3 shows the effect of the degradation of hBNP in plasma on the reactivity of different antibody pairs immobilized on latex particles with hBNP. Figure 3(A) shows the results using antibody pairs that recognize the amino acid regions of hBNP at positions 5 to 13 and 17 to 24, and Figure 3(B) shows the results using antibody pairs that recognize the amino acid regions of hBNP at positions 5 to 13 and 27 to 32. DETAILED DESCRIPTION OF THE INVENTION

[0016] <Detection method> A method for detecting human B-type natriuretic peptide (hBNP) or its precursor or cleavage products in a sample according to one aspect of the present invention comprises the step of detecting human B-type natriuretic peptide or its precursor or cleavage products in the sample by latex agglutination using a monoclonal antibody or an antigen-binding fragment thereof that recognizes the region of amino acids 17 to 24 (SEQ ID NO: 4) in the amino acid sequence shown in SEQ ID NO: 1, and a monoclonal antibody or an antigen-binding fragment thereof that recognizes the region of amino acids 5 to 13 (SEQ ID NO: 5) in the amino acid sequence shown in SEQ ID NO: 1.

[0017] The amino acid sequence shown in SEQ ID NO: 1 is the full-length amino acid sequence of hBNP, and the monoclonal antibody recognizing the region of amino acids 17 to 24 in the amino acid sequence shown in SEQ ID NO: 1 and the monoclonal antibody recognizing the region of amino acids 5 to 13 in the amino acid sequence shown in SEQ ID NO: 1 are monoclonal antibodies that specifically bind to the hBNP17-24 epitope (the region of amino acids 17 to 24 in hBNP: SEQ ID NO: 4) and the hBNP5-13 epitope (the region of amino acids 5 to 13 in hBNP: SEQ ID NO: 5), respectively. Here, "specifically" means that in a liquid system in which a protein having the above-mentioned epitope of hBNP, other protein components not having the above-mentioned epitope, and the above-mentioned monoclonal antibody are mixed, the above-mentioned monoclonal antibody does not undergo a detectable antigen-antibody reaction with other protein components, or even if some binding reaction or association reaction occurs, the reaction is clearly weaker than the antigen-antibody reaction of the above-mentioned monoclonal antibody with the protein having the above-mentioned epitope.

[0018] The monoclonal antibody recognizing the region of amino acids 17 to 24 of hBNP and the monoclonal antibody recognizing the region of amino acids 5 to 13 of hBNP can also bind to a precursor or digestion product of hBNP. The precursor of hBNP is human pro-B-type natriuretic peptide (hproBNP). The digestion product of hBNP is not particularly limited as long as it contains the region of amino acids 5 to 24 in the amino acid sequence of hBNP. The digestion product of hBNP may be, for example, a digestion product in which the 31st and 32nd amino acids have been removed from hBNP, or a digestion product in which the 1st and 2nd amino acids have been removed from hBNP.

[0019] The class of the monoclonal antibody is not limited to IgG, but may be IgY, IgM, camelid Ig, or Ig NAR. The antigen-binding fragment of the monoclonal antibody is not particularly limited, and may be Fab, Fab', F(ab')2, or a single-chain antibody (scFv).

[0020] The monoclonal antibody can be obtained, for example, by immunizing an animal with hBNP or an hBNP fragment containing the epitope using a known immunological technique, and then producing a hybridoma using cells from the immunized animal. Alternatively, the monoclonal antibody can be produced by genetic recombination techniques such as phage display and yeast display. 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.

[0021] The sample is not limited as long as it may contain hBNP or its precursor or degradation product, and may be, for example, a biological sample collected from a human or non-human animal. The biological sample may be, for example, blood (whole blood), plasma, or serum.

[0022] The step of detecting human B-type natriuretic peptide or its precursor or degradation product in a sample by latex agglutination using a monoclonal antibody or its antigen-binding fragment that recognizes the 17-24 amino acid region of hBNP and a monoclonal antibody or its antigen-binding fragment that recognizes the 5-13 amino acid region of hBNP can be carried out according to a known latex agglutination method. Specifically, first, the monoclonal antibody or its antigen-binding fragment that recognizes the 17-24 amino acid region of hBNP and the monoclonal antibody or its antigen-binding fragment that recognizes the 5-13 amino acid region of hBNP are immobilized on first and second latex particles, respectively. Next, if necessary, each antibody-immobilized latex particle is blocked with a known blocking agent such as bovine serum albumin (BSA), and then the antibody-immobilized latex particle and the sample are mixed in a buffer solution, and the mixture is incubated at a temperature at which an antigen-antibody reaction can occur. As a result, if hBNP or its precursor or degradation products are present in the sample, an antigen-antibody reaction occurs, causing the antibody-immobilized latex particles to aggregate with each other. By detecting the presence or absence of this aggregation using a known method such as light irradiation, the presence or absence of hBNP or its precursor or degradation products in the sample can be detected.

[0023] The first latex particles and the second latex particles may be the same or different, and known latex particles can be used. The materials for 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 size of the first latex particles and the second latex particles may be, for example, 0.02 to 5 μm, 0.05 to 1 μm, or 0.2 to 0.6 μm, from the viewpoint of reducing steric hindrance and obtaining sufficient detection sensitivity. In this specification, the average particle size refers to the particle size (i.e., median diameter) at which the integrated value from the smallest particle size reaches 50% of the total in a volume-based particle size distribution curve obtained by dynamic light scattering.

[0024] The first latex particles onto which a monoclonal antibody recognizing the amino acid region of hBNP at positions 17 to 24 is immobilized and the second latex particles onto which a monoclonal antibody recognizing the amino acid region of hBNP at positions 5 to 13 is immobilized may be mixed with a sample simultaneously or separately. When these antibody-immobilized latex particles are mixed with a sample separately, one of the antibody-immobilized latex particles and the sample may be mixed, and the mixture may be incubated at a temperature at which an antigen-antibody reaction can occur. After that, the other antibody-immobilized latex particles may be mixed with the mixture, and the mixture may be again incubated at a temperature at which an antigen-antibody reaction can occur.

[0025] The incubation temperature is not particularly limited and may be, for example, 20 to 37°C, and may be, in particular, 37°C, 25°C, or 20°C.

[0026] Although the epitopes recognized by the monoclonal antibody recognizing the 17-24 amino acid region of hBNP and the monoclonal antibody recognizing the 5-13 amino acid region of hBNP are close to each other, they can bind to hBNP with little influence of steric hindrance between the latex particles to which they are immobilized. Therefore, the method according to this aspect makes it possible to detect hBNP or its precursors or cleavage products by latex agglutination while suppressing the influence of steric hindrance. Furthermore, since the monoclonal antibody recognizing the 17-24 amino acid region of hBNP and the monoclonal antibody recognizing the 5-13 amino acid region of hBNP recognize regions other than the terminal region of hBNP, the method according to this aspect makes it possible to detect hBNP or its precursors or cleavage products by latex agglutination while suppressing the influence of terminal degradation of hBNP.

[0027] <Detection composition> A composition for detecting hBNP or its precursor or cleavage products according to one aspect of the present invention comprises first latex particles onto which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the region of amino acids 17 to 24 in the amino acid sequence shown in SEQ ID NO: 1, as used in the detection method according to the above aspect of the present invention, is immobilized, and second latex particles onto which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the region of amino acids 5 to 13 in the amino acid sequence shown in SEQ ID NO: 1, as used in the detection method according to the above aspect of the present invention, is immobilized. 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 cleavage products in the sample can be detected by latex agglutination.

[0028] <Detection kit> The composition according to the above aspect comprises first latex particles onto which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the amino acid region of positions 17 to 24 in the amino acid sequence shown in SEQ ID NO: 1 is immobilized, and second latex particles onto which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the amino acid region of positions 5 to 13 in the amino acid sequence shown in SEQ ID NO: 1 is immobilized. In other words, another aspect of the present invention is a kit for detecting hBNP or its precursor or cleavage products, comprising first latex particles onto which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the amino acid region of positions 17 to 24 in the amino acid sequence shown in SEQ ID NO: 1 is immobilized, and second latex particles onto which a monoclonal antibody or an antigen-binding fragment thereof that recognizes the amino acid region of positions 5 to 13 in the amino acid sequence shown in SEQ ID NO: 1 is immobilized.

[0029] The kit may further include known reagents, materials, instruments, etc., used in the latex agglutination method, such as a buffer solution. [Example]

[0030] <Test Example 1> Epitope mapping of novel antibodies Immunization of mice and rabbits Peptides 1 to 19 shown in Table 1 were synthesized. To enhance immunogenicity, each peptide was conjugated to carrier proteins such as thyroglobulin, KLH (keyhole limpet hemocyanin), OVA (ovalbumin), and BSA using various functional groups. Mice and rabbits were prepared for each peptide conjugated to a carrier protein and immunized over a two-month period at intervals of 7 to 14 days. Sigma Adjuvant System® (Merck) was used as the adjuvant. Blood samples were collected periodically during the immunization period, and antibody titers were measured by ELISA (enzyme-linked immunosorbent assay). Individuals with high antibody titers were selected, and their spleens were removed.

[0031] [Table 1]

[0032] Antibody acquisition RNA was extracted from the excised spleen using a TRIzol® Plus RNA Purification Kit (Thermo Fisher Scientific), and cDNA was obtained by reverse transcription using SuperScript® III Reverse Transcriptase (Thermo Fisher Scientific). The obtained cDNA was amplified by PCR 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 by phage display, and antibodies that specifically bind to hBNP were identified by ELISA. The sequences of various antibodies that specifically bind to hBNP were inserted into cell expression vectors and expressed in CHO (Chinese hamster ovary) cells, followed by purification using a Protein A (GE) column. Eighteen full-length antibodies were obtained, and the epitopes of all of them were identified. Most of the 18 antibodies obtained were derived from individuals immunized with peptide 19.

[0033] Epitope identification The epitopes of the obtained antibodies were confirmed as follows. First, biotinylated peptides 1–18 were synthesized (contracted to JPT Peptide Technologies) and immobilized on distinguishable beads (LumAvidin® Microspheres, manufactured by Luminex). 500 beads of each peptide-immobilized bead were added to each well of a polypropylene microplate, and one antibody was added to each well at a final concentration of 1 μg / mL. The microplate was incubated at 37°C for 1 hour, after which 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. The supernatant was removed using a magnetic stand and the microplate was washed, after which R-phycoerythrin fluorescence 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 Figure 1.

[0034] The antibody bound significantly more strongly to peptides containing the 17th and 24th amino acids of hBNP (peptides 10-17) than to peptides not containing these amino acids, suggesting that the antibody specifically recognizes the 17th to 24th amino acid region of hBNP.

[0035] <Test Example 2> Evaluation of the effect of steric hindrance Each anti-hBNP antibody solution was mixed with polystyrene beads (average particle size: 0.3 μm) and incubated overnight at 4°C to obtain antibody-immobilized beads. The antibodies used were those recognizing the 5-13 amino acid region of hBNP, those recognizing the 17-24 amino acid region of hBNP, and those recognizing the 14-20 amino acid region of hBNP. After blocking with additional BSA, beads immobilized with antibodies recognizing the 5-13 amino acid region of hBNP and those recognizing the 17-24 or 14-20 amino acid region of hBNP were suspended in buffer. Synthetic hBNP (Prospec) diluted to 10 ng / mL in PBS was added to the dilution buffer and incubated at 37°C for 5 minutes. The beads were then mixed with the antibody-immobilized bead suspension and incubated at 37°C for 5 minutes. The latex agglutination reaction was detected by irradiating with 635 nm light. The results are shown in Figure 2.

[0036] Figure 2(A) shows the results obtained using antibody pairs that recognize the amino acid regions of hBNP at positions 5 to 13 and 17 to 24, and Figure 2(B) shows the results obtained using antibody pairs that recognize the amino acid regions of hBNP at positions 5 to 13 and 14 to 20. When antibody pairs that recognize the amino acid regions of positions 5 to 13 and 17 to 24 were used, the scattering intensity of 635 nm light increased over time, whereas when antibody pairs that recognize the amino acid regions of positions 5 to 13 and 14 to 20 were used, the scattering intensity increased only slightly. This result indicates that when antibody pairs that recognize the amino acid regions of positions 5 to 13 and 17 to 24 were used, no steric hindrance occurred between the antibody-immobilized latex particles, allowing the latex reaction to proceed, whereas when antibody pairs that recognize the amino acid regions of positions 5 to 13 and 14 to 20 were used, the latex agglutination reaction was inhibited by steric hindrance. The affinities of the antibodies that recognize the 14th to 20th amino acid region and the 17th to 24th amino acid region for hBNP are 2.4 × 10 -10 M and 2.41 x 10 -10It was confirmed by Biacore (registered trademark) 8K (Cytiva) that the difference in aggregation property is not due to a difference in affinity.

[0037] <Test Example 3> Evaluation of the effect of hBNP degradation Each anti-hBNP antibody solution was mixed with polystyrene beads (average particle size: 0.3 μm) and incubated overnight at 4°C to obtain antibody-immobilized beads. The antibodies used were those recognizing the 5-13 amino acid region of hBNP, those recognizing the 17-24 amino acid region of hBNP, and those recognizing the 27-32 amino acid region of hBNP. After blocking with additional BSA, beads immobilized with antibodies recognizing the 5-13 amino acid region of hBNP and those recognizing the 17-24 or 27-32 amino acid region of hBNP were suspended in buffer. Synthetic hBNP (Prospec) diluted with hBNP-depleted plasma (Hytest) was added to the dilution buffer and incubated for 5 minutes at 37°C. The mixture was then mixed with the antibody-immobilized bead suspension and incubated for 5 minutes at 37°C. The latex agglutination reaction was detected by irradiating with 730 nm light (day 1). Furthermore, on day 1, synthetic hBNP diluted with hBNP-depleted plasma (Hytest) was left standing overnight at 4°C, and this synthetic hBNP was added to the dilution buffer and subjected to the latex agglutination reaction as described above (day 2). The results are shown in Figure 3.

[0038] In Figure 3, the scattering intensity is shown as the difference between the scattering intensity at 0 minute and the scattering intensity at 5 minutes after the start of the reaction. Figure 3(A) shows the results using an antibody pair that recognizes the amino acid regions at positions 5 to 13 and 17 to 24 of hBNP, and Figure 3(B) shows the results using an antibody pair that recognizes the amino acid regions at positions 5 to 13 and 27 to 32 of hBNP. When the antibody pair that recognizes the amino acid regions at positions 5 to 13 and 17 to 24 was used, a latex agglutination reaction occurred on both days 1 and 2, whereas when the antibody pair that recognizes the amino acid regions at positions 5 to 13 and 27 to 32 was used, a latex agglutination reaction occurred only on day 1 and no latex agglutination reaction occurred on day 2. These results indicate that hBNP stored overnight in plasma is degraded at its terminals, and that terminally degraded hBNP can be detected using antibody pairs that recognize the 5th to 13th and 17th to 24th amino acid regions, whereas terminally degraded hBNP cannot be detected using antibody pairs that recognize the 5th to 13th and 27th to 32nd amino acid regions.

Claims

1. A method for detecting human B-type natriuretic peptide or a precursor or degradation product thereof in a sample, comprising: 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; a monoclonal antibody or an antigen-binding fragment thereof that recognizes the 5th to 13th amino acid region in the amino acid sequence shown in SEQ ID NO: 1; detecting human B-type natriuretic peptide or a precursor or a degradation product thereof in a sample by latex agglutination using A method in which the precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and the degradation product of human B-type natriuretic peptide is a degradation product comprising the region of amino acids 5 to 24 in the amino acid sequence shown in SEQ ID NO:

1.

2. A composition for detecting human B-type natriuretic peptide or a precursor or degradation product thereof, comprising: first latex particles on which a monoclonal antibody or an antigen-binding fragment thereof that recognizes a region of amino acids 17 to 24 in the amino acid sequence shown in SEQ ID NO: 1 is immobilized; second latex particles onto which a monoclonal antibody or an antigen-binding fragment thereof that recognizes a region of amino acids 5 to 13 in the amino acid sequence shown in SEQ ID NO: 1 is immobilized; A composition, wherein the precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and a degradation product of human B-type natriuretic peptide is a degradation product comprising the region of amino acids 5 to 24 in the amino acid sequence shown in SEQ ID NO:

1.

3. A kit for detecting human B-type natriuretic peptide or a precursor or degradation product thereof, comprising: first latex particles on which a monoclonal antibody or an antigen-binding fragment thereof that recognizes a region of amino acids 17 to 24 in the amino acid sequence shown in SEQ ID NO: 1 is immobilized; second latex particles onto which a monoclonal antibody or an antigen-binding fragment thereof that recognizes a region of amino acids 5 to 13 in the amino acid sequence shown in SEQ ID NO: 1 is immobilized; A kit in which the precursor of human B-type natriuretic peptide is human pro-B-type natriuretic peptide, and a degradation product of human B-type natriuretic peptide is a degradation product comprising the 5th to 24th amino acid region in the amino acid sequence shown in SEQ ID NO: 1.

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