JP4423426B2 - Method of using increased concentration of adrenomedullin precursor C-terminal peptide as an indicator of cardiovascular disease or inflammatory disease - Google Patents

Description

  The present invention relates to a method for diagnosing cardiovascular disease or inflammatory disease, and a diagnostic agent or diagnostic kit therefor.

  Adrenomedullin (sometimes abbreviated as “AM”) and proadrenomedullin N-terminal 20 peptide (sometimes abbreviated as “PAMP”) are widely biosynthesized and secreted in systemic tissues. Furthermore, since the production site and the binding site are similar, AM and PAMP are likely to act as local hormones. Therefore, it is considered that the blood concentrations of AM and PAMP depend on the interrelationships such as tissue production, secretion, receptor binding, metabolism, and the like. The AM and PAMP concentrations in peripheral venous blood are thought to reflect the increase in AM and PAMP secretion in the peripheral circulation, as well as the local increase in concentration. For the measurement significance of AM and PAMP, refer to Non-Patent Document 1, for example.

  It has been shown that the concentrations of AM and PAMP in the blood increase with the severity of hypertensive / arteriosclerotic diseases, heart failure, renal failure, and inflammatory diseases. Blood AM and PAMP are associated with the severity and prognosis of these diseases. In septic shock, blood AM is markedly increased, which may have diagnostic significance. However, blood AM and PAMP concentrations are low, and the measurement method by radioimmunoassay requires complicated extraction from blood. Although direct measurement is possible using IRMA or ELISA, it is difficult to establish a measurement system because multiple types of antibodies are required.

  A precursor of human AM / PAMP (sometimes abbreviated as “ProhAM” in the present specification) is a precursor protein consisting of 185 amino acids. Although AM and PAMP are biosynthesized from ProhAM, there are many unclear points about the activity of the peptide where AM and PAMP are not biosynthesized.

ProhAM (153-185) is a peptide consisting of 33 amino acids at the C-terminal of human AM / PAMP precursor. A measurement system by radioimmunoassay (RIA) for this peptide is available as a kit from PHOENIX PHARMACEUTICAL. The company reports that blood cardiovascular concentrations are reduced in cardiovascular disease.
Non-Patent Documents 2 and 3 describe the activity of ProhAM (153-185).

  As described above, various studies have been made on ProhAM (153-185), but it is not clear about the possibility that an increase in the blood concentration of ProhAM (153-185) can be used as an index for diagnosis of a disease.

  Non-patent document 4 describes the diagnostic utility of ProhAM (45-92) in septic shock, but does not mention ProhAM (153-185).

Hormone and Clinical (0045-7167) Vol.53, No.3 Page 269-274 (2005.03) Gumusel, B., Chang, J. K., Hao, Q., Hyman, A. and Lippton, H .: Adrenotensin: an adrenomedullin gene product contracts pulmonary blood vessels. Peptides. 17: 461-465 (1996) Gumusel, B., Chang, J. K., Hyman, A. and Lippton, H .: Adrenotensin: an ADM gene product with the opposite effects of ADM. Life Sci. 57: PL87-90 (1995) Struck, J., Tao, C., Morgenthaler, N. G. and Bergmann, A .: Identification of an Adrenomedullin precursor fragment in plasma of sepsis patients. Peptides. 25: 1369-1372 (2004)

  AM and PAMP are also present in the blood, and their blood concentrations are increasing in cardiovascular diseases such as heart failure and acute myocardial infarction and septic shock. However, since both AM and PAMP quickly bind to the receptor, the blood half-life is short and the blood concentration is low, which makes it difficult to use as a disease marker.

  An object of the present invention is to provide a method for diagnosing cardiovascular disease or inflammatory disease more simply and accurately, and a diagnostic agent or diagnostic kit therefor.

  The present inventor has shown that the AM precursor (ProhAM) C-terminal peptide (ProhAM (153-185)) does not show the activity of AM and PAMP, so the blood concentration is high and the production of AM / PAMP in vivo is We focused on the possibility of reflection. And we tried to detect ProhAM (153-185) using high sensitivity radioimmunoassay (RIA). As a result, it was clarified that the ProhAM (153-185) peptide, which had only been estimated from the gene side, circulated in human blood at a high concentration. The blood concentration of ProhAM (153-185) was about 7 times higher than the blood concentration of AM, indicating the possibility of being useful as a disease marker. In heart failure, the blood concentration increased with severity. Furthermore, the concentration of ProhAM (153-185) was significantly positively correlated with the AM concentration, and the correlation coefficient was 0.67. It was also shown that there was a tendency to increase in hypertension and that it was significantly higher in hypertension with renal failure. In septic shock, blood ProhAM (153-185) was markedly increased, and it was found that diagnostic application is possible.

That is, the present invention includes the following inventions.
(1) Quantifying a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or a partial sequence thereof in a sample isolated from a subject, and increasing the amount of the polypeptide to increase cardiovascular disease or inflammation To use as an index of sexually transmitted diseases .

(2) The method according to (1),
Quantifying a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or a partial sequence thereof in a sample isolated from a subject;
Method comprising the step of comparing said amount of the polypeptide, the amount of the polypeptide isolated samples from healthy specimens of isolated sample from a subject.

(3) The method according to (1) or (2), wherein the quantification of the polypeptide is performed using an antibody that can bind to a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or a partial sequence thereof. .
(4) The antibody is an antibody derived by using, as an antigen, a polypeptide comprising the amino acid sequence of Nos. 170 to 185 of the amino acid sequence shown in SEQ ID No. 3 of the sequence listing, The method according to any one of (3).
(5) The method according to any one of (1) to (4), wherein the sample is blood.

(6) The method according to any one of (1) to (5), wherein the cardiovascular disease or inflammatory disease is heart failure, hypertension, sepsis, or septic shock.
(7) A diagnostic agent for cardiovascular disease or inflammatory disease, comprising an antibody capable of binding to a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or a partial sequence thereof.
(8) The antibody described above, wherein the antibody is an antibody derived from a polypeptide consisting of the amino acid sequence of Nos. 170 to 185 of the amino acid sequence shown in SEQ ID No. 3 of the sequence listing as an antigen. Diagnostics.

(9) A kit for diagnosing cardiovascular disease or inflammatory disease, comprising an antibody that can bind to a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or a partial sequence thereof.
(10) The antibody according to (9), wherein the antibody is an antibody derived from a polypeptide consisting of amino acid sequences Nos. 170 to 185 of the amino acid sequence shown in SEQ ID No. 3 of the sequence listing as an antigen. Diagnostic kit.

  The blood concentration of ProhAM (153-185) is higher than that of AM and PAMP and has good stability. Therefore, according to the present invention, diagnosis of cardiovascular disease or inflammatory disease is performed easily and accurately. be able to.

Subject In the present invention, the “subject” is not particularly limited as long as it is a mammal, but is preferably human.

Sample “Sample” used in the present invention specifically includes blood, urine, spinal fluid, saliva, breast milk, and tissue extract of a subject. Of these, blood is preferred. Various types of blood such as whole blood, plasma, serum, and blood cell component extracts can be used as samples.

Polypeptide The polypeptide used as a disease marker in the present invention is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or a partial sequence thereof. SEQ ID NO: 1 shows the amino acid sequence of the partial polypeptide (ProhAM (153-185)) of Nos. 153 to 185 of the precursor protein (ProhAM) of human AM / PAMP.

  In the present specification, a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or a partial sequence thereof may be referred to as “marker polypeptide”.

  The marker polypeptide is most preferably ProhAM (153-185), but is not limited thereto. The marker polypeptide may be a polypeptide containing ProhAM (153-185) as a partial sequence, and specific examples include adrenomedullin precursor, ProhAM (21-185). Further, the marker polypeptide may be a polypeptide containing a partial sequence of ProhAM (153-185), more preferably a polypeptide consisting of a partial sequence of ProhAM (153-185), specifically, ProhAM (X -185) (X is not particularly limited as long as it is an integer of 174 or less).

Method for Quantifying Polypeptide The marker polypeptide is preferably quantified by an immunological method using an antibody capable of binding to the marker polypeptide.

An antibody capable of binding to a marker polypeptide was prepared by a conventional method using a marker polypeptide, a polypeptide comprising the amino acid sequence of the marker polypeptide as a partial sequence, or a polypeptide comprising a partial sequence of the marker polypeptide as an antigen. An antibody can be preferably used. Of these, an antibody derived from a polypeptide consisting of the amino acid sequence of amino acids Nos. 170 to 185 of the amino acid sequence (ProhAM amino acid sequence) represented by SEQ ID No. 3 in the sequence listing is preferred. The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody can also be used as a fragment as long as it can bind to the marker polypeptide. Examples of antibody fragments include Fab fragments, F (ab) ′ ₂ fragments, single-chain antibodies (scFv), and the like.

  Examples of the immunological assay that can be used in the present invention include, but are not limited to, radioimmunoassay (RIA), enzyme immunoassay (ELISA), and IRMA (immunoradiometric assay). Of these, the radioimmunoassay method is preferred.

A monoclonal antibody can be prepared, for example, by the following procedure.
The above-mentioned antigen is administered to animals together with itself, a carrier, or a diluent at a site where antibody production is possible by administration of the antigen. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Examples of the animal to be used include mammals such as monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, etc., but mice and rats are preferred. The antibody titer in the antiserum can be measured by a conventional method.

  By selecting individuals with antibody titers from animal mice immunized with antigen, collecting spleen or lymph nodes 2 to 5 days after the final immunization, and fusing the antibody-producing cells contained therein with myeloma cells A monoclonal antibody-producing hybridoma can be prepared. The fusion operation can be performed according to a known method, for example, the method described in Nature 256: 495 (1975). Examples of the fusion promoter include polyethylene glycol (PEG). Examples of myeloma cells include NS-1, P3U1, SP2 / 0, and the like.

  The selection of the monoclonal antibody can be performed according to a known method or a method similar thereto, but can usually be performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium may be used as long as the hybridoma can grow. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the antiserum.

  The separation and purification of the monoclonal antibody is the same as the separation and purification of a normal polyclonal antibody, for example, a salting-out method, an alcohol precipitation method, an isoelectric point precipitation method, an electrophoresis method, an adsorption / desorption method using an ion exchanger (eg, DEAE), It can be carried out according to an ultracentrifugation method, a gel filtration method, an antigen-binding solid phase or a specific purification method using protein A or protein G, and the like, according to an immunoglobulin separation and purification method.

On the other hand, a polyclonal antibody can be prepared, for example, by the following procedure.
Polyclonal antibodies are prepared by, for example, producing a complex of an antigen and a carrier, immunizing a mammal in the same manner as the above-described monoclonal antibody production method, collecting an antibody-containing substance against active haptoglobin from the immunized animal, It can manufacture by performing isolation | separation purification. The antigen used for the production of the polyclonal antibody is the same as that for the production of the monoclonal antibody. When forming a complex of an antigen and a carrier, the type of carrier and the mixing ratio of the antigen and the carrier can be any ratio and any ratio as long as the antibody can be efficiently produced against the antigen cross-linked to the carrier. It may be crosslinked with Examples of the carrier include bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, and the like. Various condensing agents can be used for coupling the antigen and the carrier, and active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, dithiobilidyl group, and the like are used.

  The complex of an antigen and a carrier is administered to a immunized animal at a site where antibody production is possible, together with the carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration can usually be carried out about once every 2 to 6 weeks, about 3 to 10 times in total. Examples of animals used include mammals similar to those used in the production of monoclonal antibodies. Polyclonal antibodies can be collected from blood, ascites, etc., preferably blood of animals immunized by the above method. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed by the same procedure as the separation and purification of the monoclonal antibody.

Diseases to be diagnosed Diseases diagnosed by the present invention are cardiovascular diseases or inflammatory diseases, and specifically include heart failure, hypertension, sepsis, or septic shock. It is particularly effective in diagnosing hypertension associated with impaired renal function among hypertension. In addition, since the blood concentration of the marker polypeptide of the present invention is positively correlated with the blood concentration of adrenomedullin, the method of the present invention can be used for diseases associated with increased blood concentration of adrenomedullin (specifically, pulmonary hypertension). Disease, diabetes, bronchitis, pregnancy, arteriosclerosis, myocardial infarction).

Diagnosis In the present invention, cardiovascular disease or inflammatory disease is diagnosed using an increase in the amount of marker polypeptide in the test sample as an index.

  In the present invention, “diagnosis” refers to determining whether a subject suffers from a cardiovascular disease or inflammatory disease, and whether there is a risk of suffering from a cardiovascular disease or inflammatory disease in the future. Determination of whether or not, determination of severity of cardiovascular or inflammatory disease, determination of therapeutic effect, and determination of whether there is a risk of recurrence of cardiovascular or inflammatory disease after treatment It is a concept.

  Particularly in heart failure, since the amount of marker polypeptide varies depending on the severity, the method of the present invention is useful for determining the severity of heart failure.

  Moreover, in the case of hypertension, it can be determined by the method of the present invention whether or not hypertension is accompanied by renal dysfunction.

  When determining whether or not a subject suffers from cardiovascular disease or inflammatory disease, the amount of marker polypeptide in the sample of a healthy sample can be used as a reference. In determining the effect of treatment, the amount of marker polypeptide in a sample collected from a subject before treatment may be used as a reference value.

Diagnostic Agent or Diagnostic Kit Containing Antibody The present invention also relates to a diagnostic agent for cardiovascular disease or inflammatory disease comprising an antibody capable of binding to a marker polypeptide.

  The diagnostic agent of the present invention can be made into a kit together with necessary reagents. For example, a radioimmunoassay kit includes a radiolabeled tracer peptide.

  The kit may further contain a standard sample, a buffer solution, a lysis solution, a washing solution, a reaction stop solution, an instruction manual, and the like.

  An antibody capable of binding to the marker polypeptide may be affixed on the chip at a high density to form a protein chip, and such a protein chip is also included in the diagnostic kit of the present invention.

Experimental operation
Preparation of anti-ProhAM (170-185) antibody (Preparation of C-terminal recognition antibody of ProhAM (153-185))
ProhAM (170-185) (amino acid sequence AHGAPAPPSGSAPHFL) and bovine thyroglobulin were conjugated (condensed) by the glutaraldehyde method and immunized. ProhAM (170-185) (8 mg) and bovine thyroglobulin (10 mg) were dissolved in 2 ml of 0.1 M phosphate buffer ( _PH 7.4). Under stirring at 0 ° C., 100 μl of 5% glutaraldehyde was added and reacted for 10 minutes. After dialyzing against physiological saline and 0.05M phosphate buffer ( _PH 7.4, containing 0.08M NaCl), it was adjusted to 11 ml with the same phosphate buffer, and NaN ₃ was added to 0.1% and stored frozen. For immunization, the conjugate of ProhAM (170-185) and bovine thyroglobulin is mixed with Titer Max Gold (Sigma-Aldrich) adjuvant in an equivalent amount, and sufficiently emulsified. And then injected subcutaneously. The booster immunization was performed every two weeks for the first half of the dose from one month later. Blood samples were collected 7 days after the booster immunization, and after serum separation by centrifugation, NaN ₃ was added to a concentration of 0.1% and stored frozen.

Examination of antibody titer The antibody titer was examined using a tracer labeled with ¹²⁵ I. Under the same conditions as RIA, only the antiserum concentration was changed, and the antiserum concentration capable of binding 50% tracer was determined and compared. Rabbits whose antibody titers did not rise were immunized about 3 times, and those that continued to rise were immunized continuously. It was possible to collect up to about 60 ml at a time from the ear artery, and those with high titers were repeatedly collected.

Establishment of radioimmunoassay system
(1) Preparation of tracer Radioiodine label of peptide (N-tyr-ProhAM (170-185), amino acid sequence: YAHGAPAPPSGSAPHFL) has been reported (Kitamura K, Matsui E, Kato J, Katoh F, Kita T, Tsuji T, Kangawa K, Eto T. Adrenomedullin (11-26): a novel inherent hypertensive peptide isolated from bovine adrenal medulla. Peptides. 2001 Nov; 22 (11): 1713-8. To 25 μl of 0.4 M sodium acetate buffer ( _PH 5.6), peptide (5 μg), NaI ( ¹²⁵ I) 1 mci / 10 μl, lactoperoxidase 20 ng, H ₂ O ₂ 200 ng (added in two portions), 37 ° C., The reaction was allowed for 20 minutes.

The tracer was purified by changing the CH ₃ CN concentration by reverse phase HPLC. Column, TSK ODS 120A (4.6 x 150 mm); flow rate, 1.0 ml / min; solvent system, linear gradient elution from A: B = 80: 20 to A: B = O: 100 (80 min). H ₂ 0: CH ₃ CN: 10% TFA = 90: 10: 1 (v / v); (B) H ₂ 0: CH ₃ CN: 10% TFA = 40: 60: 1 (v / v).
The purified tracer was added with BSA at 0.25% to 0.5%, dispensed, and stored frozen at −20 ° C.

(2) Radioimmunoassay method
RIA has been reported (Kitamura K, Matsui E, Kato J, Katoh F, Kita T, Tsuji T, Kangawa K, Eto T. Adrenomedullin (11-26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla. Peptides. 2001 Nov ; 22 (11): 1713-8.). The RIA for a standard buffer (hereinafter abbreviated as RIA buffer) using BSA 0.25% in 0.05M sodium phosphate _{(PH 7.4), NaCl 0.08M,} EDTA-2Na 0.025M, the solution was added N _a N ₃ 0.05% . As the RIA tube, an acrylic tube (10 × 78 mm, Central Trade) was used.

The total RIA reaction solution is 400 μl, and is composed of the following solutions.
200μl: Standard peptide solution or unknown sample solution
100 μl: Tracer labeled with ¹²⁵ I (17500-18500 cpm)
100 μl: Antiserum (4 times the final dilution)
(All of the above are dissolved and diluted in RIA buffer.)

The above three solutions were sufficiently stirred and then incubated at 4 ° C. for 20 hours. Add 100 μl of 1% bovine γ-globulin solution (0.05M phosphate buffer, dissolved in _PH 7.4-0.08M NaCl) and stir, then add 1 ml of 23% polyethylene glycol (dissolved in the same buffer) to form a precipitate. The reaction was stopped by. The mixture was allowed to stand at 4 ° C. for 15 minutes, centrifuged at 4 ° C. and 3000 rpm for 30 minutes, the supernatant was aspirated, and the radioactivity of the pellet portion was measured with a γ-counter. Samples were usually assayed in duplicate.

(3) Antiserum dilution and radioimmunoassay standard Antiserum dilution was performed in exactly the same manner as the antibody titer measurement. A dilution curve was prepared by changing the antiserum concentration only under the same conditions as RIA and measuring the radioactivity bound to the antibody. At the same time, a dilution curve is prepared even in a system in which an antigen peptide is excessively added, and the antiserum concentration is determined so that the difference in the amount of radioactivity bound depending on the presence or absence of the antigen is sufficiently large and the dilution is maximized. Used for.

  Synthetic peptides were quantified by amino acid analysis and used as RIA standards. Based on the peptide concentration, 12 points were prepared in a total of 12 points: 512 fmol / 200 μl, 256 fmol / 200 μl to 0.5 fmol / 200 μl, two-fold diluted 10 points, 0 fmol / 200 μl, and used as actual standards. Similarly, various peptide standards for examining cross-activity were also adjusted to a maximum of 1 μg / 100 μl. In the case of RIA of the tissue extract, a considerable amount of Tris and acetic acid are contained in the sample solution. In this case, the standard was prepared with a buffer having the same composition as the sample.

Experimental result
(1) Examination of cross-activity in radioimmunoassay The cross-activity of various peptides in this radioimmunoassay system was examined. As a result, there is 100% cross-activity with ProhAM (153-185) and N-tyr-ProhAM C-II (170-185), and 0% cross-activity with ProhAM (153-167), adrenomedullin and PAMP. It was confirmed that. These results indicate that the prepared antiserum contains an antibody that recognizes the C-terminus of ProhAM.

FIG. 1 shows a standard curve of a radioimmunoassay of ProhAM (153-185). The midpoint is 42 fmol / tube, and measurement is possible up to 4 fmol / tube.
The intra-assay error was 3.5%, and the inter-assay error was 10%.

(2) Additive recovery test
When 4, 8, 32 fmol of ProhAM (153-185) was added and the measurement was performed, the recovery rate was 86-118%, which was good.

(3) Dilution test Radioimmunoassay was performed using 100 μl, 50 μl, or 25 μl of two types of plasma samples (plasma samples from heart failure patients and healthy individuals), and the amount of plasma sample used (corresponding to the dilution ratio) The correlation with the detected amount of ProhAM (153-185) was confirmed. The results are shown in FIG. In FIG. 2, white circles are data from plasma samples from heart failure patients, and black circles are data from plasma samples from healthy individuals. As shown in FIG. 2, almost good linearity was obtained.

(4) Stability of ProhAM (153-185) It was stable for 8 hours from sampling to freezing of plasma separation.
It was confirmed that there was no significant change in the value even when freeze-thaw was repeated up to 4 times.
In cryopreservation, it was stable for more than 6 months.

(5) Qualitative analysis of peptides in plasma by HPLC
108 ml of healthy human plasma was desalted and deproteinized using SEPPAC (recovery rate: about 60%). Analyzed by HPLC. The analysis conditions are as follows.
Column: 050412 HPLC RP chemcosorb 7ODSH 0x250mm
Eluent: (A) :( B) = 100: 0 ~ 0: 100, linear gradient 2.0ml / fr,
(A) H ₂ 0: CH ₃ CN: 10% TFA = 90: 10: 1 (v / v); (B) H ₂ 0: CH ₃ CN: 10% TFA = 40: 60: 1 (v / v )

  The recovery rate by HPLC was about 50%. The result of HPLC is shown in FIG. In FIG. 3, the horizontal axis represents the elution time (minutes) and the vertical axis represents the peptide amount. The fraction indicated by the arrow in FIG. 3 includes proAM (153-185). From this result, it was shown that most of ProhAM C-terminal partial peptide ProhAM (X-185) that binds to the antiserum used in this experiment is ProAM (153-185).

(6) Blood concentration of ProhAM (153-185) in heart failure patients The plasma concentration of ProAM (153-185) in heart failure patients (NYHA) was compared with that of healthy individuals.
Heart failure patients (NYHA) were classified into three stages (NYHA I-II, NYHA III, NYHA IV). The results are shown in Table 1 and FIG.

  As shown in Table 1 and FIG. 4, it was confirmed that the blood concentration of ProhAM (153-185) was significantly higher in patients with severe heart failure.

For comparison, AMT (AM total) concentration in blood was measured by a conventional measurement method on samples from the same subject. This method is described in detail in Ohta H, Tsuji T, Asai S, Tanizaki S, Sasakura K, Teraoka H, Kitamura K, Kangawa K. A simple immunoradiometric assay for measuring the entire molecules of adrenomedullin in human plasma.Clin Chim Acta. 1999 Sep; 287 (1-2): 131-43. This method is a method for measuring the blood concentration of adrenomedullin by an immunoradiometoric assay (IRMA) using an antibody that recognizes the ring structure of adrenomedullin and an intermediate part connecting the ring and the C-terminal. There are two forms of adrenomedullin contained in living organisms (AM-gly and AM-CONH ₂ ), but “AMT” includes both AM-gly and AM-CONH ₂ . Therefore, in this specification, “AMT detection value (or concentration)” means the total detection value (or concentration) of AM-gly and AM-CONH ₂ . The measurement results are shown in Table 2 and FIG.

  Comparing the two methods confirmed that ProhAM (153-185) is more preferred as a marker for patients with heart failure.

(7) Correlation between ProhAM (153-185) and AMT Plotting the AMT detection value in healthy subjects and patients with heart failure on the horizontal axis and the ProhAM (153-185) detection value on the vertical axis confirmed the correlation (FIG. 6). The correlation coefficient was 0.670.

(8) Blood concentration of ProhAM (153-185) in patients with hypertension The concentration of ProAM (153-185) in plasma of patients with hypertension was compared with that of healthy individuals.
Hypertensive patients were classified into two stages (hypertensive patients without organ damage and hypertensive patients with renal dysfunction). The results are shown in Table 3 and FIG.

  As shown in Table 3 and FIG. 7, it was confirmed that the blood concentration of ProhAM (153-185) was significantly higher in hypertensive patients with renal dysfunction.

(9) ProhAM (153-185) blood concentration in patients with septic shock ProAM (153-185) concentration in plasma of patients with severe sepsis / septic shock was compared with that in healthy individuals. For comparison, the AMT concentration was similarly measured. The results are shown in Table 4 (ProAM (153-185) detection value), Table 5 (AMT detection value) and FIG. In FIG. 8, the black column shows the AMT detection value, and the white column shows the ProAM (153-185) detection value.

  As shown in Tables 4 and 5 and FIG. 8, it was confirmed that the blood concentration of ProhAM (153-185) was significantly higher in patients with severe sepsis / septic shock. In addition, ProhAM (153-185) was confirmed to be more preferable as a marker for severe sepsis / septic shock compared to AMT.

1 shows a standard curve of a radioimmunoassay for ProhAM (153-185). The correlation between the amount of plasma sample used (corresponding to the dilution factor) and the detected amount of ProhAM (153-185) is shown. White circles are data from plasma samples from patients with heart failure, and black circles are data from plasma samples from healthy individuals. The analysis result by HPLC of the peptide in a healthy subject plasma sample is shown. The blood levels of ProAM (153-185) in healthy individuals and patients with three-stage heart failure (NYHA) are shown. The blood concentrations of AMT in healthy subjects and patients with three-stage heart failure (NYHA) are shown. The correlation between the AMT blood level and the ProhAM (153-185) blood level in healthy individuals and patients with heart failure is shown. The blood levels of ProhAM (153-185) in healthy individuals and patients with two stages of cardiac hypertension are shown. The ProhAM (153-185) blood level (white) and the AMT blood level (black) are shown in healthy subjects and patients with severe sepsis / septic shock.

Claims (11)

Isolated sample from a subject, consisting of SEQ ID NO: 1 amino acid sequence or a partial sequence shown in the sequence listing to quantify polypeptides of the polypeptide amounts cardiovascular disease or an inflammatory disorder an increase in The method used as an indicator. The method of claim 1, comprising:
Quantifying a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or a partial sequence thereof in a sample isolated from a subject;
Comparing the amount of the polypeptide in a sample isolated from a subject with the amount of the polypeptide in a sample isolated from a healthy sample. The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing in a sample isolated from the subject using an antibody that can bind to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or a partial sequence thereof Alternatively, a method comprising quantifying a polypeptide containing a partial sequence thereof and using an increase in the amount of the polypeptide as an indicator of cardiovascular disease or inflammatory disease . The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing in a sample isolated from the subject using an antibody that can bind to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 Alternatively, a method comprising quantifying a polypeptide containing a partial sequence thereof and using an increase in the amount of the polypeptide as an indicator of cardiovascular disease or inflammatory disease,
Quantifying a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or a partial sequence thereof in a sample isolated from a subject using the antibody;
Comparing the amount of the polypeptide in a sample isolated from a subject with the amount of the polypeptide in a sample isolated from a healthy sample. 5. The antibody according to claim 3 or 4 , wherein the antibody is an antibody derived from a polypeptide consisting of the amino acid sequence of Nos. 170 to 185 of the amino acid sequence shown in SEQ ID No. 3 of the Sequence Listing as an antigen. the method of. The method according to any one of claims 1 to 5 , wherein the sample is blood. The method according to any one of claims 1 to 6 , wherein the cardiovascular disease or inflammatory disease is heart failure, hypertension, sepsis, or septic shock. Comprising an antibody capable of binding to a polypeptide comprising the amino acid sequence or a partial sequence thereof shown in SEQ ID NO: 1, a diagnostic agent for cardiovascular disease or inflammatory disease. The diagnostic agent according to claim 8 , wherein the antibody is an antibody derived from a polypeptide consisting of the amino acid sequence of Nos. 170 to 185 of the amino acid sequence shown in SEQ ID No. 3 of the Sequence Listing as an antigen. . Comprising an antibody capable of binding to a polypeptide comprising the amino acid sequence or a partial sequence thereof shown in SEQ ID NO: 1, a diagnostic kit of cardiovascular disease or an inflammatory disease. The diagnostic antibody according to claim 10 , wherein the antibody is an antibody derived by using, as an antigen, a polypeptide consisting of amino acid sequences Nos. 170 to 185 of the amino acid sequence shown in SEQ ID No. 3 of the Sequence Listing. kit.

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