JP2015137272A - Novel peptide and method for detecting disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel peptide associated with human B-type natriuretic peptide (hBNP).SOLUTION: The peptide has the following characteristics of: (I) being detected by sandwich assay using antibodies which bind to hBNP but not bind to hproBNP and hBNP (3-32), and antibodies which bind to C-terminal tail of hBNP; and (II) having tolerance rather than hBNP to the hBNP decomposition mechanism in blood. A disease using increase and decrease in hBNP as an indicator, such as heart disease, is detected by measuring the peptide present in a sample.

Description

  The present invention relates to novel peptides related to human B-type natriuretic peptide.

  Since BNP was first isolated from porcine brain (Non-Patent Document 1), it was named Brain Natriuretic Peptide (BNP). However, in humans, BNP is suggested to be synthesized and secreted in the heart (Non-patent document 2), and isolated and identified from the heart (Non-patent document 3). Thus, the word brain has been changed to type B, and now BNP means type B natriuretic peptide.

  BNP circulates in the blood and exerts strong cardiovascular and renal effects. Human B-type natriuretic peptide (hereinafter referred to as hBNP) consists of 32 amino acid residues and corresponds to amino acid sequence 77-108 of human pro-B-type natriuretic peptide (hereinafter referred to as hproBNP) which is a precursor of hBNP. . hBNP consists of a 17-amino acid cyclic moiety closed by a disulfide bond between two cysteine residues, a 9-amino acid N-terminal tail, and a 6-amino acid C-terminal tail. In addition, it is known that when hBNP is incubated in blood, the N-terminal two amino acid residues are eliminated and hBNP (3-32) is generated (Non-patent Document 4). hBNP (3-32) is a peptide corresponding to amino acid sequence positions 3-32 of hBNP and corresponding to positions 79-108 of the amino acid sequence of hproBNP. Furthermore, not only hBNP and hBNP (3-32) but also hBNP (4-32) and hBNP (5-32) (respectively positions 4-32 and 5-32 of the amino acid sequence of hBNP are present in the blood of heart disease patients. It has been confirmed that there are various molecules derived from hproBNP, such as a peptide corresponding to position 32 (Patent Document 1).

  Currently, the recognition sites of the two anti-hBNP antibodies used in the sandwich assay are the hBNP circular portion and the C-terminal tail, respectively. When such a sandwich assay is used, not only hBNP but also all molecules having the same cyclic part and C-terminal tail as hBNP are detected without distinction (hereinafter, the generic name of molecules detected by the sandwich assay is referred to as hBNP molecule). Group).

International Publication No. 2010/023749 Pamphlet

Nature 332, 78-81 (1988) Biochem. Biophys. Res. Commun. 159, 1427-1434 (1989) FEBS Lett. 259, 341-345 (1990) Clin Chim Acta 316: 129-135 (2002)

  The object of the present invention is to provide a novel peptide related to hBNP.

As a result of intensive studies on the above problems, the present inventors have completed the present invention. That is, the present invention is as follows.
(1) A peptide having the following characteristics.
(I) It is detected by a sandwich assay using an antibody that does not bind to hproBNP and hBNP (3-32) but binds to hBNP and an antibody that binds to the C-terminal tail of hBNP. And (II) more resistant to hBNP degradation mechanisms in blood than hBNP.
(2) A method for detecting a disease using as an index the increase or decrease in hBNP, comprising measuring the peptide according to (1) present in a specimen.

  The present invention is described in detail below. The peptides of the present invention have been found in blood samples of several heart disease patients. The peptide is detected by sandwich assay using an antibody that does not bind to hproBNP and hBNP (3-32) but binds to hBNP (hereinafter referred to as hBNP-specific antibody) and an antibody that binds to the C-terminal tail of hBNP. Peptide. Further, the peptide has a characteristic that it is more resistant to hBNP degradation mechanism in blood than hBNP, that is, it is not easily degraded.

  When the hBNP molecule group concentration and hBNP concentration (detected by a sandwich assay using an antibody binding to the C-terminal tail of hBNP and hBNP) in the blood sample of a heart disease patient were measured, It has been found that blood samples having a high hBNP concentration ratio are classified into two groups: blood samples and small blood samples (hereinafter referred to as group 1 and group 2, respectively). Initially, the ratio of the hBNP concentration to the hBNP molecule group concentration in Group 1 was considered to be because the hBNP degradation activity in the blood sample was reduced, and the residual amount of hBNP was large. Therefore, when commercially available hBNP was added to a blood sample of a heart disease patient and the hBNP concentration was measured after a certain period of time, the decrease in hBNP concentration was not significantly different between Group 1 and Group 2. That is, it was found that the hBNP degrading activity of the group 1 blood samples was not particularly reduced.

  Therefore, the conventional hBNP is present in the blood sample of group 2, but the novel peptide of the present invention is present in the blood sample of group 1 in addition to the conventional hBNP, and it is considered that the value was increased accordingly. .

  Since this peptide of the present invention can be detected by a sandwich assay using an hBNP-specific antibody and an antibody that binds to the C-terminal tail of hBNP, it was considered to be hBNP itself. However, since hBNP is very easily decomposed in blood, the peptide of the present invention is hardly decomposed in blood. Therefore, it is considered that it is not a hBNP itself but a subtype of hBNP.

  The present invention further provides a method for detecting a disease by measuring the peptide present in a sample and using hBNP increase / decrease as an index. Examples of the disease using increase / decrease in hBNP as an index include hypertension, heart disease, kidney disease and the like, and are expected to be particularly useful for heart disease. The approximate peptide may be measured by various methods such as ELISA and mass spectrometry. Examples of the specimen include blood.

  The present invention is a novel peptide related to BNP. The present invention is expected to be useful for diagnosing diseases such as hypertension, cardiac function / kidney function, and the like, where hBNP increases and decreases.

2 is a graph showing the results of Example 1. 6 is a graph showing the results of Example 2. 10 is a graph showing the results of Example 3.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by a present Example.

[Example 1] Isolation of hBNP-specific antibody An hBNP-specific antibody was isolated by the following method.

(1) Preparation of immunoconjugate solution The peptide consisting of the amino acid sequence described in SEQ ID NO: 1 was obtained by requesting peptide synthesis from a trader. The peptide fragment solution (10 mg / ml, dissolved in phosphate buffered saline (PBS)) and maleimide-activated KLH solution (10 mg / ml, dissolved in PBS) were mixed at a volume ratio of 1: 1. The reaction was allowed to stir at room temperature for 2 hours. Then, the conjugate solution for immunization was obtained by dialyzing the reaction solution against PBS for 2 days at 4 ° C.

(2) Immunization The above-described immunization conjugate solution (1 mg / ml) and Freund's complete adjuvant are mixed in equal amounts to form an emulsion, which is 0.1 ml in the abdominal cavity of a mouse (female, 6 weeks old). Injections were made 4 times at weekly intervals. The dose of conjugate per mouse was 50 μg. Further, one week after the final immunization, a immunization conjugate solution diluted to 0.5 mg / ml with PBS (0.1 ml) was administered intraperitoneally as a booster.

(3) Cell fusion Three days after the booster immunization, the spleen of the mouse was removed, and the cells were destroyed with erythrocyte lysis buffer. The remaining cells were suspended in E-RDF medium and used as spleen lymphocytes used for cell fusion. Next, 0.4 × 10 ⁷ myeloma cells similarly suspended in E-RDF medium were mixed with 1.6 × 10 ⁷ splenic lymphocytes, centrifuged (1000 rpm, 5 minutes), and the supernatant was removed. The cell precipitate was suspended in mannitol buffer (250 mM mannitol, 100 mM calcium chloride, 100 mM magnesium chloride), and electric cell fusion was performed. The cell suspension subjected to the electric cell fusion is suspended in 200 ml of HAT medium (GIT medium containing 100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine), and is suspended in each well of 10 384-well cell culture plates. A 0.05 ml aliquot was dispensed. Hybridoma growth was observed after about 7 days.

(4) Screening Antibody was obtained from the obtained hybridoma by a conventional method. The antibody was immobilized on an ELISA plate at 0.1 μg / well and blocked with 1 wt% skim milk. After washing the wells, hBNP (Sigma-Aldrich, Brain Natural Peptide-32 human) was adjusted to a concentration of 1 ng / mL, and 100 μL of the solution was added to the wells for reaction. After washing the well, an alkaline phosphatase labeled product of antibody BC23-11 (Japanese Patent Laid-Open No. 2011-122957) that recognizes the C-terminal tail of hBNP was added to the well and allowed to react. After the reaction, the binding between the antibody and hBNP was observed by measuring the alkaline phosphatase activity according to a conventional method. The above-mentioned measurement was performed using hproBNP (HyTest, Human recombinant proBNP glycosylated) or hBNP (3-32) (obtained by peptide synthesis by a vendor) instead of hBNP. Observed using the method. The results are shown in FIG. It was revealed that the antibody does not bind to hproBNP and hBNP (3-32) but binds to hBNP (hBNP specific antibody).

[Example 2] Measurement of hBNP molecule group and hBNP Using a commercially available fully automatic enzyme immunoassay device (AIA-2000, manufactured by Tosoh Corporation) as an immunoassay device, hBNP molecule group and hBNP were measured by a one-step sandwich method. . For the measurement of the hBNP molecule group, a commercially available immunoassay reagent E test “TOSOH” II (BNP) (manufactured by Tosoh Corporation) was used. For the measurement of hBNP, the hBNP-specific antibody obtained in Example 1 and an immunoassay reagent using BC23-11 were used. More specifically, the immunoassay reagent includes hBNP-specific antibody immobilized on a water-insoluble carrier and BC23-11 labeled with alkaline phosphatase.

  A blood sample (that passed 90 minutes or more at room temperature after blood collection) was added to the immunoassay reagent, and alkaline phosphatase activity was measured with the above-mentioned apparatus according to a conventional method. In addition, in the sandwich immunoassay described above, if a standard curve based on the activity intensity and hBNP concentration is prepared in advance using a standard product containing a known concentration of hBNP, the activity intensity of an unknown sample of hBNP can be measured. Thus, the hBNP concentration can be calculated from the standard curve.

  FIG. 2 shows the results of measuring the hBNP molecule group concentration and hBNP concentration of each blood sample. It was found that the blood sample (group 1) and the blood sample (group 2) having a large ratio of hBNP concentration to hBNP molecule group concentration are classified into two groups.

[Example 3] Measurement of hBNP-degrading activity of blood sample Three blood samples were randomly selected from the two groups classified in Example 2, respectively. 42 μl of 20 ng / ml hBNP (Sigma-Aldrich, Brain Natural Peptide-32 human) was added to 800 μl of each blood sample. After 0 minutes, 30 minutes, 60 minutes, and 90 minutes after the addition, the hBNP concentration was measured by the same immunoassay as in Example 2. The results are shown in FIG. Similarly for all blood samples, the hBNP concentration decreased with time. That is, it was found that there was no difference in hBNP degradation activity between blood samples. That is, it is considered that the hBNP in the blood sample of Group 1 includes not only conventional hBNP but also a subtype of hBNP that is difficult to be decomposed in blood.

Claims (2)

A peptide having the following characteristics.
(I) an antibody that does not bind to a peptide corresponding to amino acid sequence 3-32 of human pro-B-type natriuretic peptide and human B-type natriuretic peptide but binds to human B-type natriuretic peptide, and human B-type natriuretic It is detected in a sandwich assay using an antibody that binds to the C-terminal tail of the peptide. And (II) more resistant to the degradation mechanism of human B-type natriuretic peptide in blood than human B-type natriuretic peptide. A method for detecting a disease using as an index the increase or decrease in human B-type natriuretic peptide, comprising measuring the peptide according to claim 1 present in a specimen.

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