WO2008012941A1 - Method for diagnosis of heart failure - Google Patents

Abstract

Disclosed is a method for the diagnosis of heart failure at an early stage. Specifically, disclosed is a method for the determination of the degree and/or prognosis of heart failure, characterized by measuring a PTX3 level in a sample by using an anti-PTX3 monoclonal antibody.

Description

 Specification

 How to diagnose heart failure

 Technical field

 [0001] The present invention relates to a method for detecting PTX 3 in blood with high sensitivity and determining the degree of heart failure.

 Background art

 [0002] PTX3, also known as Pentraxin, Pentaxin, TSG-14, M PTX 3, was found to be expressed in interleukin 1 (IL_1) stimulated human umbilical cord endothelial cells It is a secreted protein belonging to the Pentraxin family (Non-patent Document 1).

 [0003] The Pentraxin family includes Cre a c ti i v e p ro t e i n (and RP) and se r u amy l o i p P cmp o ne t (S A P), which are known as inflammatory proteins. Pentraxin is also known as L o n g Pen rt a x i n, and CRP is also called S h rt P ent r a x i n. Pentraxin is a name because it has a CRP sequence portion in its structure, and it is assumed that it functions as an inflammatory protein. Unlike CRP and SAP, PTX3 is not guided by I L-6. In addition, PTX 3 protein-expressing cell types are also different from CRP and SAP, suggesting that PTX 3 also has functions different from CRP and SAP (Non-patent Documents 2 and 3).

 [0004] On the other hand, in patients with acute myocardial infarction, which is considered to be a kind of inflammatory reaction, the blood concentration of PTX 3 increases, and it can be an indicator of small vessel inflammation. The detection by this etc. was discovered, and the involvement in inflammation was estimated (nonpatent literature 4).

 [0005] The term “inflammation” covers a wide range, including dermatitis and inflammation of various organs. Among them, inflammation of blood vessels leads to serious diseases such as heart disease and brain disease.

According to the data of 2006 of the “Demographic Statistics” conducted by the Ministry of Health, Labor and Welfare every year. As a result, the death toll from Japanese heart disease is as high as 15.5%, which is known to be the second leading cause of death. Furthermore, a detailed examination of the causes of death from heart disease reveals that heart failure and acute myocardial infarction are common, each of which accounts for 5.0% and 4.3% of the cause of death, and the number of deaths due to heart failure is extremely high. I understand that. In addition, the prognosis of critically ill patients is very poor, and the 1-year mortality rate ranges from 50% to 60% for IV heart failure according to the NYHA classification.

 Diagnostic methods for heart failure include: (1) cardiac catheterization, coronary angiography, myocardial biopsy, (2) nuclear medicine, ultrafast CT, MR I, (3) electrocardiogram, (4) chest x-ray, (5 ) Echocardiography, (6) Measurement of blood norepinephrine and renin, (7) Measurement of blood BN P, etc. Diagnosis ■ Currently, monitoring is being conducted.

 (1) Cardiac catheterization, coronary angiography, biopsy of myocardium: large invasion

 (2) Nuclear medicine examination, ultra-high speed CT, MR I: Less invasive but less information available.

 (3) ECG: Ischemia is important for the diagnosis of arrhythmia, but there is little information on heart failure itself.

 (4) Chest X-ray: Useful in determining the extent of cardiac enlargement and pulmonary congestion, but difficult to distinguish when present such as pneumonia.

 (5) Echocardiogram: Structure ■ Functional aspects can be analyzed. In particular, in ischemic heart disease, it is possible to capture local movement abnormalities non-invasively, but there are body types that are difficult to photograph as well as requiring skill in judgment. It is also difficult to detect heart failure due to diastolic failure.

 (6) Norepinephrine and renin in blood: Effective in severe cases, but it is difficult to distinguish in mild cases.

(7) Blood BN P: Not only heart failure but also high in atrial fibrillation, hypertension, decreased renal function, anemia, cirrhosis, heart failure in obesity, chronic obstructive pulmonary disease, chronic nephritis non-dialysis patients The diagnosis rate is low. Therefore, there is a demand for the development of a measurement method that is not only invasive and simple, but also excellent in measurement sensitivity and specificity, which can replace the above-described diagnostic method.

[0007] As a conventional method for measuring PTX 3, a method based on the ELISA method described in Non-Patent Document 3 is known. In this assay, PTX 3 concentrations range from 0.5 ng / ml before myocardial infarction due to coronary artery anomaly, which is an ischemic heart disease, to a maximum of 22 ng L after 7.5 hours. And then suddenly decreased, the coronary artery seems to have no abnormality in the blood concentration of normal people is described to be reduced to 0.5 to 2.5 ng / mL, but measurement sensitivity In addition to being unsatisfactory, there is no mention of whether or not the degree of heart failure can be determined.

 [0008] In addition, the present inventor previously developed a new PTX 3 measurement system and measured the blood PTX 3 concentration, thereby positioning it as a reserve group for myocardial infarction, which is an ischemic heart disease. We have successfully diagnosed a patient with unstable angina, but have succeeded in developing a drug for measuring mild heart disease caused by abnormal coronary arteries and measuring diseases caused by abnormalities other than coronary arteries. (Patent Document 1).

 Non-licensed literature 1 ： B re V I o r o o e t a I.: J. B i o l. C hem., 267 (3 1), 22 1 90— 7 (1 992)

 Non-Patent Document 2: J. Biol. Chem., 267 (3 1), 22 1 90—7 (1 992)

 Unlicensed Document 3: Dom a k u K o (A r t e r i o s c l e r o s i s), 24 (7 -8), 375-80 (1 996)

 Non-Patent Document 4: Circua latio, 10 02, 636— 4 1 (2000) Patent Document 1: WO 2005-08098 1

 Disclosure of the invention

 Problems to be solved by the invention

 An object of the present invention is to provide a method for diagnosing mild to severe heart failure and determining the degree and / or prognosis of heart failure.

Means for solving the problem [0010] In the present invention, for the purpose of providing a method for determining the degree and / or prognosis of heart failure, blood PTX 3 concentration is measured using an anti-PTX 3 monoclonal antibody, As a result of comparison with the degree of symptoms of heart failure patients, a clear correlation was found between the degree of heart failure and PTX 3 concentration, and by measuring PTX 3 concentration, the degree of heart failure could be determined. The present inventors have found a relationship between prognosis and PTX 3 concentration and found that an accurate treatment guideline can be determined for each patient, thereby completing the present invention.

 [001 1] That is, the present invention provides a method for determining the degree of heart failure and the prognosis of heart failure, characterized by measuring the concentration of PTX3 protein in a test sample.

 [0012] The present invention also provides a diagnostic agent for the degree of heart failure and the prognosis of heart failure, which comprises an anti-PTX 3 monoclonal antibody.

[0013] The present invention also relates to P in a test sample using an anti-P T X3 monoclonal antibody.

It provides a diagnostic agent for the degree and / or prognosis of heart failure characterized by measuring T X 3 concentration.

[0014] The present invention also provides use of an anti-PTX 3 monoclonal antibody for the manufacture of a diagnostic agent for the degree of heart failure and the prognosis of heart failure.

 The invention's effect

 [0015] According to the present invention, since the degree from mild heart failure to severe heart failure can be accurately diagnosed, it is possible to determine a treatment guideline for early treatment and prevention of transition to severe heart failure. it can.

 Brief Description of Drawings

 [0016] [Fig. 1] shows the results of immunostaining of PTX3 in the myocardium of patients with idiopathic dilated cardiomyopathy

FIG. 2 shows the measurement results of blood PTX 3 in patients with non-ischemic chronic heart failure and in the control group.

[Fig. 3] Measurement results of PTX 3 in blood of heart failure patients in each group of NYHA classification [Fig. 4] Shows the ratio of blood PTX 3 high concentration patients (4.4 ng / mL) in each group of NY HA classification.

 [FIG. 5] shows the results of Ka p I a n—Me i e r angle analysis of c a d i a c e v nt in patients with high blood PTX 3 concentration at Yamagata University Hospital.

 [Fig. 6] shows the results of q u ar t i I e analysis of blood P T X 3 levels that cause c a r d i a c e v e n t in heart failure patients at Yamagata University Hospital.

 FIG. 7 shows the survival curve (no event) and log rank test results of the PTX 3 concentration high value group and low value group in Example 6 according to the force plan Meyer method.

 FIG. 8 shows the results of gel filtration of recombinant タ ン パ ク 質 3 protein prepared in Example 11 and ΡΤΧ3 protein in clinical samples (Example 12).

 FIG. 9 shows the reactivity of anti-antibody X3 monoclonal antibody with full-length X3 in a Western blot under reducing / non-reducing conditions.

 [FIG. 10] The results of Western blotting using the expression of Ρ Τ X 3 polypeptide in Example 18 and 血清 を X 3 sensitized mouse serum as the primary antibody in Example 18 are shown. Α and A ': Samples obtained by treating full length PTX 3 under reducing and non-reducing conditions, respectively. B and B ': Samples of PTX3 N-terminal polypeptide (N— PTX3 (2)) treated under reducing and non-reducing conditions, respectively. C and C ′: Samples of PTX3 C-terminal polypeptide (C—PTX3) treated under reducing and non-reducing conditions, respectively. D and D ′: Samples obtained by treating CHO cell culture supernatant lysate without gene transfer under reducing and non-reducing conditions (negative control), respectively.

FIG. 11 shows the result of Western blotting using the culture supernatant of Hypridoma strain Hy b — 3423 that produces anti-HB s antibody in Example 18. A and A ′: Samples obtained by treating full length PTX 3 under reducing and non-reducing conditions, respectively. B and B ′: Samples of PTX 3 N-terminal polypeptide (N— PTX3 (2)) treated under reducing and non-reducing conditions, respectively. C and C ': Samples of Ding 3 treated with O-end polypeptide (C-PTX3) under reducing and non-reducing conditions, respectively. D and D ': CHO cell culture supernatant lysate without gene transfer Samples treated under reducing and non-reducing conditions (negative control), respectively.

FIG. 12 shows the results of Western plotting using anti-PTX3 monoclonal antibody P PMX 01 04 in Reference Example 11. A and A ′: Samples obtained by treating full length PTX3 under reducing and non-reducing conditions, respectively. B and B ′: Samples of PTX 3 N-terminal polypeptide (N— PTX3 (2)) treated under reducing and non-reducing conditions, respectively. C and C ′: Samples of PTX3 C-terminal polypeptide (C_PTX3) treated under reducing and non-reducing conditions, respectively. D and D ': Samples of CHO cell culture supernatant lysate without gene transfer treated under reducing and non-reducing conditions (negative control), respectively.

 FIG. 13 shows the results of separation of full-length PTX3 digests by reverse-phase HPLC in Example 19;

 FIG. 14 shows the reactivity of the lysylated peptidase digested with P PMX 01 04 after reduction treatment of full-length P TX 3 of Example 19 as a result of the E L ISA method.

 FIG. 15 shows the reactivity of lysyl peptidase digested with P PMX 01 05 after reduction treatment of full-length P T X 3 of Example 19 as a result of the E L I S A method.

 FIG. 16 shows the results of Example 20, in which full-length P TX 3 was digested with lysyl peptidase under non-reducing conditions, electrophoresed with SDS_PAGE, and stained with Kumashi-Puriant Blue. 1: molecular weight marker, 2: molecular weight marker, 3: undigested full length PTX3, 4: digestion after 0 hours, 5, digestion after 0.5 hours, 6: digestion after 1 hour, 7: digestion after 2 hours, 8: digestion 4 hours later, 9: 8 hours after digestion.

 FIG. 17 shows the relationship between the lysendopeptide digestion time under non-reducing conditions of full length Ρ Τ X 3 in Example 20 and the reactivity with ΡΜΧ ΡΜΧ 01 04.

 FIG. 18 shows the relationship between the ligation peptide digestion time under non-reducing conditions of full length Ρ Τ X 3 in Example 20 and the reactivity with ΡΜΧ ΡΜΧ 01 05.

 [Fig. 19] A comparison of standard curves of the conventional ELISA kit and the present ELISA kit is shown.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, the measurement includes quantitative or non-quantitative measurement, for example, Non-quantitative measurements include simply measuring whether or not PTX 3 protein is present, measuring whether or not PTX 3 protein is present above a certain amount, and determining the amount of PTX 3 protein to other samples (eg, Measurement and comparison with a control sample), and quantitative measurement includes measurement of the concentration of PTX 3 protein, measurement of the amount of PTX 3 protein, and the like. The base sequence of the PTX 3 gene is shown in SEQ ID NO: 1, and the amino acid sequence of PTX 3 is shown in SEQ ID NO: 2.

 [0018] The test sample is not particularly limited as long as it may contain PTX 3 protein, but a sample collected from the body of an organism such as a mammal is preferred, and more preferably from a human. It is a collected sample. Specific examples of the test sample include blood, interstitial fluid, plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph fluid, saliva, urine, and the like. Preferred are blood, serum, and plasma. A sample obtained from a test sample such as a culture solution of cells collected from the body of an organism is also included in the test sample of the present invention.

 [001 9] Heart failure begins with subjective symptoms such as tiredness and shortness of breath, and is a serious illness that leads to death as the whole body develops congestive blood via depressive blood in the lungs, kidneys, and extinguisher.

The “degree of heart failure” can be based on the degree of heart function performed by the New York Heart Association according to the NYHA classification. The NYHA classification evaluates the severity of heart failure by ability to move. A degree I indicates that the patient has heart disease but is not restricted in subjective motor skills. Level II is due to heart disease, and there are some restrictions on subjective motor ability. With normal exercise, it shows symptoms such as fatigue, dyspnea, palpitation, and angina pain. The third degree is due to heart disease, there is a significant limitation of exercise capacity, usually less than a mild exercise symptoms. At the most severe degree IV, there is a symptom even at rest, and even the lightest exercise can cause an exacerbation of the symptom. The assessment of heart failure according to the NYHA classification reflects a decline in exercise capacity due to a decline in cardiac function. Therefore, limiting mild exercise means more serious heart failure. Heart failure is classified by etiology and is broadly divided into ischemic chronic heart failure and non-ischemic chronic heart failure. Of these, the most common cause of non-ischemic chronic heart failure is dilated cardiomyopathy, followed by valvular disease. Other causes include myocarditis or myocardial disease.

 [0020] "Assessment of the degree of heart failure" has a positive correlation between the Ding 3 concentration and the YHA classification, as shown in the examples below. As the PTX 3 concentration increases, the heart failure becomes severe. Specifically, PTX3 concentration 2.2 ± 1. 1 ng / mL or less does not lead to heart disease (0 degree); PTX 3 concentration 3.4 soil 2.2 ng / ml ^ iNYHA I degree, PTX 3 concentration 4.4 ± 2. 9 ng / mL is NYHA II degree, PTX 3 concentration 6.8 ± 4. 8 ng / mL is NY HA III degree, 1 5. 7 ± 9. 9 ng / m L is preferably determined as NYHAIV degree and the degree of heart failure.

 “Determining the prognosis of the degree of heart failure patients” means monitoring the symptoms of patients with heart failure, that is, determining the extent of heart failure after (prognosis) of patients diagnosed with heart failure. Furthermore, in the prognosis of a patient diagnosed with heart failure, if the patient's PTX 3 concentration is determined to be 4 ng / mL or higher, preferably 4.4 ng ZmL or higher, readmission or death ( It is preferable to determine that there is a risk of a cadiac event.

 In the method of the present invention, the PTX 3 protein is preferably measured by an immunological assay using an anti-PTX3 antibody. Hereinafter, the measurement method using the anti-PTX 3 antibody will be described in detail.

[0022] <1. Preparation of anti-PTX3 antibody>

The anti-PTX 3 antibody used in the present invention only needs to specifically bind to the PTX 3 protein. Preferred is an antibody that exhibits a high binding affinity for the three-dimensional structure of PTX 3, more preferably a high binding affinity for the three-dimensional structure of PTX 3, and that does not cross-react with CRP or SAP. More preferably, P PMX01 02 (FERM B P-1 0326), P PMX01 04 (FERM BP-1 07 1 9) and P PMX01 05 (FERM BP— 1 0720), most preferably P PMX01 04 (FERM B P- 1 07 1 9) and P PMX01 05 (FERM BP— 1 0720) .

 The hybridomas of P PMX01 02, P PMX01 04 and P PMX 01 05 are FERM BP-1 0326, FERM BP-1 07 1 9 and FERM BP-1 0720, respectively. ： Deposited at Tsukuba City East, Ibaraki Pref., 1st 1st 1st 1st 6th (P PMX01 02: Date of deposit: February 15, 2005, FE RM BP— 1 07 1 9 and FE RM BP _ 1 0720: Date of deposit: September 22, 2005 (Heisei 17).

 [0023] In more detail, PTX 3 is a protein composed of a single-chain polypeptide having a total length of 381 amino acids (SEQ ID NO: 2) (hereinafter, this protein is also referred to as PTX3 protein or full-length PTX3). . Amino acid numbers 1 to 17 are signal peptides, which are cleaved during the process of secretion to the mature PTX 3 protein. Therefore, the N-terminal part of PTX3 protein is amino acid numbers 18 to 1 78 (hereinafter referred to as the 1 \ 1 terminal part of Ding 3). The C-terminal part is amino acid numbers 1 79 to 381 (hereinafter referred to as PTX). This is a polypeptide composed of 3 C-terminal sites. This C-terminal region is called a pentraxin domain and is highly homologous to CRP and S AP belonging to the pentraxin family.

 [0024] The origin, type (monoclonal, polyclonal) and shape of the antibody are not limited. Specifically, known antibodies such as mouse antibodies, rat antibodies, human antibodies, chimeric antibodies, humanized antibodies can be used. The antibody may be a polyclonal antibody, but is preferably a monoclonal antibody.

 In addition, the anti-PTX3 antibody immobilized on the support and the anti-PTX3 antibody labeled with a labeling substance may recognize the same epitope of the PTX3 molecule, but preferably recognize different epitopes.

[0025] The anti-PTX 3 antibody used in the present invention is a polyclonal antibody using known means. Or as a monoclonal antibody. As the anti-PTX 3 antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Mammal-derived monoclonal antibodies include those produced by a hybridoma and those produced by a host transformed with an expression vector containing an antibody gene by genetic engineering techniques.

[0026] Monoclonal antibody-producing hybridomas can be basically produced using known techniques as follows. In other words, using PTX 3 as a sensitizing antigen, this is immunized according to a normal immunization method, and the resulting immune cells are fused with a known parent cell by a normal cell fusion method. It can be produced by screening monoclonal antibody-producing cells.

 [0027] Specifically, a monoclonal antibody can be prepared as follows.

 First, PTX3 used as a sensitizing antigen for antibody acquisition is obtained by purifying from the culture supernatant of available cells. Alternatively, it can be obtained according to the method disclosed in Special Table 2 0 0 2-5 0 3 6 4 2.

 Next, this purified PTX3 protein is used as a sensitizing antigen. Alternatively, a partial peptide of PTX3 can be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human PTX 3, or a part of the PTX 3 gene can be incorporated into the expression vector, and further, natural PTX 3 Can also be obtained by proteolytic degradation. The portion and size of PTX3 used as a partial peptide are not limited, but are preferably partial peptides having a C-terminal portion.

 [0028] The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Rodent animals such as mice, rats, hamsters, etc., rabbits and monkeys are used.

[0029] Immunization of an animal with a sensitizing antigen is performed according to a known method. For example, a common method is to inject a sensitizing antigen intraperitoneally or subcutaneously in a mammal. Is done. Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Sline) or physiological saline, etc., and then mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if desired. After emulsification, administer to mammals several times every 4 to 2 days. An appropriate carrier can also be used during immunization with the sensitizing antigen. In particular, when a partial peptide having a small molecular weight is used as a sensitizing antigen, it is desirable to immunize albumin or keyhole limpet by binding to a carrier protein such as mosocyanin.

 [0030] After immunizing a mammal and confirming that the desired antibody level rises in serum, immune cells are collected from the mammal and subjected to cell fusion. Preferred immune cells In particular, splenocytes are mentioned.

 [0031] Mammalian myeloma cells are used as the other parent cell to be fused with the immune cells. This myeoma cell is known in various known cell lines such as P 3 (P 3 x 63 A g 8.653) (J. I mm no I. (1 979) 1 23, 1 548-1 550 ) P 3 x 63 A g 8 U. 1 (Current Topics in Microbiology and I mm uno I ogy (1 978) 8 1, 1 _7), N S_ 1 (Ko hler. G. and Milstein, C. E ur. J. I mm unol. (1 976) 6, 5 1 1 — 5 1 9) MP C- 1 1 (Marguelie s. DH eta and C el I (1 976) 8, 405— 4 1 5 ), SP 2/0 (Shu I man, M. eta, Nature (1 978) 276, 269-270), FO (de St. Groth, S. F. eta I., J. I mm uno to Methods (1 980) 35, 1 -2 1) S 1 94 (Trow bridge, I. S. J. E x p. M e d. (1 978) 1 48, 3 1 3- 323), R 2 10 (Galfre, G. eta, Nature (1 979) 277, 1 3 1-1 33) and the like are preferably used.

[0032] The cell fusion between the immune cells and myeloma cells is basically performed by a known method such as the method of Kohler and Milstein et al. (Ko h Ier. G. and M i I stein, C., M ethods E nz ymo can be performed according to (198 1) 73, 3-46) etc.

 [0033] More specifically, the cell fusion is performed in a normal nutrient culture medium in the presence of, for example, a cell fusion promoter. For example, polyethylene glycol (P EG), Sendai virus (HV J) or the like is used as a fusion promoter, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency as desired. .

 [0034] The use ratio of immune cells and myeloma cells can be arbitrarily set.

 For example, the number of immune cells is preferably 1 to 10 times that of myeloma cells. As the culture medium used for the cell fusion, for example, RPM I 1 640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, and other normal culture liquids used for this kind of cell culture can be used. In addition, serum supplements such as fetal calf serum (FCS) can be used in combination.

 [0035] In cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and pre-warmed to about 37 ° C in polyethylene glycol (PEG).

(For example, an average molecular weight of about 1 000 to 6000) A solution is usually added at a concentration of 30 to 60% (w / v) and mixed to form a desired fused cell (hybridoma). Subsequently, an appropriate culture solution is sequentially added, and the operation of centrifuging and removing the supernatant is repeated to remove cell fusion agents and the like which are not preferable for the growth of the hybridoma.

 [0036] The hybridoma thus obtained is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing with the above HAT culture solution is continued for a sufficient period of time (usually several days to several weeks) to kill cells (non-fusion cells) other than the target hybridoma. Next, the usual limiting dilution method is performed, and screening and single cloning of the hybridoma producing the target antibody are performed.

[0037] Screening and single cloning of the antibody of interest can be achieved by using known antigens. A screening method based on an antibody reaction may be performed. For example, the antigen is bound to a carrier such as beads made of polystyrene or a commercially available 96-well microtiter plate, reacted with the culture supernatant of the hybridoma, the carrier is washed, and then the enzyme-labeled secondary antibody is used. By reacting, it can be determined whether or not the target antibody reacting with the sensitizing antigen is contained in the culture supernatant. Hybridomas producing the desired antibody can be cloned by limiting dilution or the like. In this case, the antigen used for immunization may be used.

 In addition to immunizing animals other than humans to obtain the above-mentioned hybridomas, human lymphocytes are sensitized to PTX3 in vitro, and the sensitized lymphocytes are fused with human-derived myeloma cells having the ability to divide, It is also possible to obtain a desired human antibody having a binding activity to PTX 3 (see Japanese Patent Publication No. 1-59878). Furthermore, PTX 3 as an antigen is administered to a transgenic animal having all repertoires of human antibody genes to obtain anti-PTX 3 antibody-producing cells, and human antibodies against PTX 3 are obtained from the immortalized cells. OK (see WO 94/25585 pamphlet, WO 93/1 2227 pamphlet, WO 92/0391 8 pamphlet, WO 94/02602 pamphlet).

 [0038] The hybridoma producing the monoclonal antibody produced as described above can be subcultured in a normal culture solution, and can be stored in liquid nitrogen for a long period of time.

 [0039] To obtain a monoclonal antibody from the hybridoma, a method of culturing the hybridoma according to a normal method and obtaining it as a culture supernatant, or transferring the hybridoma to a mammal compatible therewith. For example, it can be administered and proliferated to obtain ascites. The former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.

 [0040] A method may be used in which genes encoding these antibody fragments are constructed, introduced into an expression vector, and then expressed in an appropriate host cell.

[0041] These antibodies are all of the protein encoded by the PTX 3 gene. As long as the characteristic of recognizing the length or part is not lost, a low molecular weight antibody such as an antibody fragment (fragment) or a modified antibody may be used. Specific examples of antibody fragments include, for example, Fab, Fab ′, F (ab ′) 2, Fv, Diabody, and the like. Such antibody fragments can be obtained by digesting the Fc portion of IgG with pepsinpapain, constructing genes encoding these antibody fragments, introducing them into expression vectors, (Eg, Co, MS eta I., J. Immuno (1 994) 1 52, 2968-2976; Better, M. and Horwitz, AH, Methods E nz ymo on (1 9

89) 1 78, 476-496; PI uckthun, A. and Skerra, A., Methods E nz ymo (1 989) 1 78, 4 97— 5 1 5; Lamoyi, E., Methods E nz ymo I. (1 986) 1 2 1, 652-663; Ro usseaux, J. Eta I

, M e t h o d s E n z ymo to (1 986) 1 2 1, 663— 66

9; Bird, R.E.and Walkele, B.W., TrendsBiotec hno (see (1 99 1) 9, 1 32-1 37).

The antibody produced as described above can be isolated from cells and host animals and purified to homogeneity. Separation and purification of the antibody used in the present invention can be carried out using an affinity column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose FF (manufactured by GE Healthcare) and the like can be mentioned. In addition, separation and purification methods used for ordinary proteins may be used, and the method is not limited at all. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns other than the above-mentioned affinity column, filters, ultrafiltration, salting out, dialysis and the like (Antibodies AL aboratory Manual. E d H arl ow, D avid L ane, C old S pring H arbor L aboratory, 1 9 88) [0043] As a modified antibody, an anti-PTX 3 antibody bound to various molecules such as a labeling substance can also be used. The “antibody” in the present invention includes these modified antibodies. Such a modified antibody can be obtained by chemically modifying the obtained antibody. Antibody modification methods have already been established in this field.

 [0044] <Measurement of 2 Ding 3>

 PTX 3 measured in the present invention is not particularly limited, and may be full-length PTX3 or a fragment thereof.

 The method for detecting PTX3 protein contained in the test sample is not particularly limited, but it is preferably detected by an immunological method using an anti-PTX3 antibody. Examples of immunological methods include Radioimmunoassay, Enzymimmunoassay, Fluorescent Imunoassay, Luminescent Immunonossay, Immunoprecipitation, Immunoturbidimetric Method, Western Plot, Immunostaining, Immunodiffusion Method, etc. Enzyme-imnoassay is preferred, and particularly preferred is the enzyme-free “5” free enzyme-enhanced Mi method (ELI SA) (eg, san dw ich ELI SA). The above-described immunological methods such as ELISA can be performed by methods known to those skilled in the art.

 [0045] As a general detection method using an anti-PTX 3 antibody, for example, an anti-PTX 3 antibody is immobilized on a support, a test sample is added thereto, and the mixture is incubated and then anti-PTX 3 antibody and PTX 3 protein are used. A method of detecting PTX 3 protein in a test sample by washing after binding and detecting PTX 3 protein bound to the support via the anti-PTX 3 antibody can be mentioned.

[0046] Examples of the support used in the present invention include insoluble polysaccharides such as agarose and cellulose, synthetic resins such as silicone resin, polystyrene resin, polyacrylamide resin, nylon resin, and polycarbonate resin. And an insoluble support such as glass. These supports can be used in the form of beads or plates. For beads, these Can be used. In the case of a plate, a multi-well plate (96-well multi-well plate, etc.) or a single chip of biosensor can be used. The anti-PTX 3 antibody can be bound to the support by a commonly used method such as chemical bonding or physical adsorption. All of these supports can be commercially available.

 [0047] The binding between the anti-PTX3 antibody and the PTX3 protein is usually performed in a buffer solution. As the buffer solution, for example, phosphate buffer solution, Tris buffer solution, citrate buffer solution, borate buffer solution, carbonate buffer solution and the like are used. Incubation is performed under conditions that are already in use, for example, incubation at 4 ° C to room temperature for 1 to 24 hours. Washing after incubation is not particularly limited as long as it does not interfere with the binding between the PTX3 protein and the anti-PTX3 antibody. For example, a buffer containing a surfactant such as Tween20 is used.

 [0048] In the method for measuring PTX3 protein of the present invention, a control sample may be installed in addition to the test sample for detecting PTX3 protein. Control samples include negative control samples that do not contain PTX3 protein and positive control samples that contain PTX3 protein. In this case, detect the PTX 3 protein in the test sample by comparing the result obtained with the negative control sample without PTX 3 protein and the result obtained with the positive control sample containing PTX 3 protein. Is possible. In addition, a series of control samples with varying concentrations are prepared, detection results for each control sample are obtained as numerical values, a standard curve is created, and based on the standard curve from the values of the test sample It is also possible to quantitatively detect the PTX 3 protein contained in the test sample.

 [0049] As a preferred embodiment of the measurement of the PTX3 protein bound to the support via the anti-PTX3 antibody, a method using an anti-PX3 antibody labeled with a labeling substance can be mentioned.

[0050] For example, the test sample is brought into contact with the anti-PTX 3 antibody immobilized on the support and washed. Later, detection is performed using a labeled antibody that specifically recognizes the PTX 3 protein.

[0051] Labeling of the anti-ΡX3 antibody can be performed by a generally known method. As the labeling substance, a labeling substance known to those skilled in the art, such as a fluorescent dye, an enzyme, a coenzyme, a chemiluminescent substance, and a radioactive substance, can be used. (3 2 Ρ, 1 4 C, 1 2 5 1, 3 H, 1 3 1 I, etc.), fluorescein, rhodamine, danssil mouth lid, umbelliferone, luciferase, peroxidase, alkaline phosphatase S-galactosidase, β-glucosidase, horseradish / monoxidase, glucoamylase, lysozyme, saccharide doxidase, microperoxidase, biotin and the like. When using biotin as the labeling substance, it is preferable to add avidin to which an enzyme such as alkaline phosphatase is bound after adding the biotin-labeled antibody. A known method such as glutaraldehyde method, maleimide method, pyridyl disulfide method, periodate method, or the like can be used for the binding of the labeling substance and the anti-antibody antibody.

 [0052] Examples of enzyme labeling methods for antibodies include, but are not limited to, the hinge method and the non-hinge method. The hinge method uses a thiol group generated by reducing the disulfide bond in the part called the hinge part between the F (ab ′) 2 part that has the antigen-binding ability of antibody I g G. It is a method of binding the enzyme molecule with '. On the other hand, the non-hinge method does not specify which reactive group of the antibody is used, but in many cases, the amino group of the antibody is used to bind the antibody molecule and the enzyme molecule.

[0053] Specifically, a solution containing an anti-PTX 3 antibody is added to a support such as a plate, and the anti-PTX 3 antibody is fixed to the support. After washing the plate, block it with BSA, gelatin, albumin, etc. to prevent non-specific binding of proteins. Wash again and add the test sample to the plate. After incubation, wash and add labeled anti-PTX 3 antibody. After moderate incubation, the plate is washed and the labeled anti-PTX 3 antibody remaining on the plate is detected. Inspection For example, in the case of labeling with a radioactive substance, it can be detected by liquid scintillation or RIA. In the case of labeling with an enzyme, a substrate is added, and enzymatic changes of the substrate, for example, color development, can be detected with an absorptiometer. Specific examples of substrates include 2, 2_azinobis (3_ethyl benzothiazoline mono-6-sulfonic acid) diammonium dium salt (ABTS), 1, 2_ phenylene diamine (ortho phenylene diamamine) , 3, 3 ', 5, 5'-tetramethylbenzidine (TMB). In the case of a fluorescent substance, it can be detected by a fluorometer.

 [0054] As a particularly preferred embodiment of the method for measuring PTX 3 protein of the present invention, the Fc portion unrelated to the antigen-binding ability of antibody IgG is removed, and the method described in Example 9 for the enzyme labeling method of the measured antibody And a method using an antibody labeled with

[0055] Specifically, a solution containing an anti-PTX3 antibody is added to a support such as a plate to immobilize the anti-PTX3 antibody. After washing the plate, block with BSA, for example, to prevent nonspecific binding of proteins. Wash again and add the test sample to the plate. After incubation, wash and add peroxidase directly labeled anti-PTX3 antibody. After moderate incubation, the plate is washed, a substrate corresponding to the enzyme is added, and PT X3 protein is detected using the enzymatic change of the substrate as an indicator.

 [0056] As another embodiment of the method for measuring PTX3 protein of the present invention, one or more types of primary antibodies that specifically recognize PTX3 protein, and one type of secondary antibody that specifically recognizes the primary antibody. The method used above can be mentioned.

[0057] For example, one or more types of anti-PTX 3 antibodies immobilized on a support are brought into contact with a test sample, incubated, washed, and PTX 3 protein bound after washing is treated with primary anti-PTX 3 Detection is performed with an antibody and one or more secondary antibodies that specifically recognize the primary antibody. In this case, the secondary antibody is preferably labeled with a labeling substance. [0058] As another embodiment of the method for measuring PTX 3 protein of the present invention, a detection method using an agglutination reaction can be mentioned. In this method, ΤX3 can be detected using a carrier sensitized with an anti-ΡΡ3 antibody. As the carrier for sensitizing the antibody, any carrier may be used as long as it is insoluble, does not cause a non-specific reaction, and is stable. For example, latex particles, bentonite, collodion, kaolin, fixed sheep erythrocytes and the like can be used, but it is preferable to use latex particles. As the latex particles, for example, polystyrene latex particles, styrene monobutadiene copolymer latex particles, polyvinyl toluene latex particles, and the like can be used, and it is preferable to use polystyrene latex particles. Mix the sensitized particles with the sample and stir for a period of time. The higher the concentration of anti-ΡΡΤ3 antibody in the sample, the greater the degree of particle aggregation. Therefore, ΡΡX3 can be detected by observing the aggregation with the naked eye. It is also possible to detect turbidity due to aggregation by measuring with a spectrophotometer or the like.

 [0059] Another embodiment of the method for measuring ΡΡX3 protein of the present invention includes, for example, a method using a biosensor utilizing a surface plasmon resonance phenomenon. A biosensor using the surface plasmon resonance phenomenon can observe a protein-protein interaction as a surface plasmon resonance signal in real time without using a minute amount of protein and labeling. For example, it is possible to detect the binding between PTX3 protein and anti-PTX3 antibody by using a biosensor such as BI Acore (manufactured by Pharmacia). Specifically, a test sample is brought into contact with a sensor chip on which an anti-PTX3 antibody is immobilized, and the PTX3 protein that binds to the anti-PTX3 antibody can be detected as a change in resonance signal.

 [0060] The measurement method of the present invention can be automated using various automatic inspection apparatuses, and a large number of samples can be inspected at a time.

[0061] The present invention is also aimed at providing a diagnostic agent for use in determining the degree and / or prognosis of heart failure, and the diagnostic agent preferably contains an anti-PTX 3 antibody. here Diagnostic agents include kits. When the diagnostic agent is based on the ELISA method, it may contain a carrier for immobilizing the antibody, and the antibody may be bound to the carrier in advance. When the diagnostic agent is based on an agglutination method using a carrier such as latex, a carrier to which an antibody is adsorbed may be included. In addition, the diagnostic agent may appropriately contain a blocking solution, a reaction solution, a reaction stop solution, a reagent for treating the sample, and the like.

 Example

 [0062] Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.

 <Example 1 Construction and measurement of ELISA system>

 In order to detect PTX3 protein in blood, a PTX3 sandwich ELISA system was constructed as follows. That is, F (ab ') 2 PPPMX 0 1 04 (FE RM BP— 1 07 1 9) is applied to the antibody coated on the 96 well plate at 5 g / mL, ^ 00 UL / we II, 4 The solution was solidified by incubating at 1 ° C.

6 1 Iの洗浄緩衝液 （0. 05% ( v/v) Tw e e n 20, P B S) で 3回洗浄後、 A B I社のィムノアッセィスタビライザー (A B I # 1 0— 60 1 — 00 1 ) を 1 50 L加え、 ブロッキングを行 つた。 室温で数時間後、 あるいは 4°Cで一晩保管後、 精製蛋白質、 ヒト血清 などを、 動物血清などを含む希釈緩衝液 （50mM T r i s _C I p H 8. 0, 0. 1 5M N a C I ) で適当に希釈したものを加え 2時間室温で インキュベートした。 次いで、 動物血清などを含む P B S (-) で [0064] Next day 300

6 After washing 3 times with 1 I wash buffer (0.05% (v / v) Tween 20, PBS), use ABI Immunoassay Stabilizer (ABI # 1 0— 60 1 — 00 1). 1 50 L was added and blocking was performed. After several hours at room temperature or after overnight storage at 4 ° C, add purified protein, human serum, etc. to a dilution buffer containing animal serum (50 mM Tris_C I pH 8. 0, 0. 1 5M N a CI) was diluted appropriately and incubated at room temperature for 2 hours. Next, with PBS (-) containing animal serum etc.

Incubated and one Bok at room temperature for 2 hours added HRPO diluted such that L (horseradish peroxide O Kishida one peptidase) labeled _{F ab} 'of P PMX 0 1 05 antibody. After discarding the reaction solution, it was washed 5 times with 300; UL / _we II wash buffer, and then developed using Scytek TMB (Cat #TM 49 9 9) according to the attached protocol. The absorbance was measured with a microplate reader. To convert the concentration of PTX 3 protein in the sample, use the spreadsheet software GI a Analysis was carried out using phPadPRISM (GlaphPadsoftwareInc. ver. 3.0).

 <Example 2> Immunostaining of PTX 3 in the myocardium of patients with idiopathic dilated cardiomyopathy> Myocardial tissue obtained by biopsy from patients with idiopathic dilated cardiomyopathy waiting for heart transplantation with informed consent The presence or absence of PTX 3 was confirmed. The myocardial tissue was immunostained after fixation with normal paraffin fixation (primary antibody: PPMX0102, secondary antibody: mouse monoclonal I g G). For immunostaining, P PMX 01 02 (FERM BP-1 0 326) was used as an anti-PTX3 antibody. For the staining of corn tr, the primary antibody P PMX 01 02 was not used, but the secondary antibody mouse monoclonal IgG was used. As a result, it was revealed that the cytoplasm was clearly diffused in comparison with the control (Fig. 1).

 <Example 3 Measurement of blood PTX 3 in patients with non-ischemic chronic heart failure> To elucidate the relationship between disease and plasma PTX 3 concentration in patients with non-ischemic chronic heart failure, 98 healthy subjects And 43 patients with non-ischemic chronic heart failure (NYHA classification: groups I, II, III, and IV), using plasma samples collected from veins by EDTA, using the ELI 3 8 method shown in Example 1. The concentration of 乂 3 was measured. The time of blood collection was; such as immediately before the introduction of S-blocker, the heart failure was stable, and there was no a c t i v e infection.

 As a result, it was 2.1 2 ± 1.03 ng / mL in the healthy group, whereas it was 4.1 4 ± 2.74 ng / mL in patients with non-ischemic chronic heart failure (Fig. 2).

 <Example 4> Measurement of blood P T X 3 in each group of heart failure patients N Y H A classification>

The subjects were 44 patients with heart failure who visited Yamagata University Hospital between August 2002 and March 2005 (hereinafter referred to as the non-exclusion group). Twenty-five people with no heart failure (14 men, 1 woman, age 66.0 ± 13.4 years) were used as controls. The age and sex of the control group were matched to be proportional to the heart failure patient group. The severity of heart failure is determined by NYHA (New York Heart Association)! ~ Classified according to IV category one. To elucidate the relationship between heart failure patients and plasma PTX 3 concentration, 25 subjects without heart failure (contro group I), NYHAI group 27 in NYHA classification, NY HA II group 51, NY HA III Concentration measurement was performed according to the PTX 3 measurement method described in Example 1 above, using plasma samples collected from the vein of 52 people in the group and 4 people in the NY HA IV group 1 from 4 people.

 Diagnosis of heart failure is made by a cardiologist by dyspnea or pulmonary congestion, peripheral edema, left ventricular hypertrophy or chest x-ray, echocardiography, or radiocardiography abnormalities It was.

 To examine the relationship between plasma PTX 3 concentration and the severity of heart failure according to the NYHA classification, we used S tat V iew V er. 5.0 (SAS institute I n c. As a result, the mean value of the contro I group was 2.2 ± 1.1 ng / ml, the average value of the NY HA I group was 3.4 ± 2.2 ng / ml, and the mean value of the NY HA II group was 4. 4 ± 2. 9 ng / mL, NY HA III group average is 6.8 ± 4.8 ng / mL, NYHAIV group average is 15.7 ± 9.9 ng / mL Control group vs NYHAII group, NY HA III group and NYHAIV group, NY HA I group and NYHAII group vs. NYHAIII group and NYHAIV group, and also significant difference between NY HA III group and NY HA IV group (p <0. 0 1) was observed, and an increase in the mean value was confirmed in proportion to the severity of heart failure (Fig. 3).

In addition, 144 heart failure patients were observed prospectively with the occurrence of cardiac event as an end point (observation period: mean 546 ± 327 days, renci 5-1177). Here, cardiacevent refers to death or readmission due to worsening heart failure. The incidence of cardiac events was 22.2%, 29.4%, 63.5%, and 92.9% in the NY HA I, NY HA II, NYHA III, and NY HA IV groups, respectively. (Figure 4). The more severe the heart failure according to the NYHA classification, the higher the incidence of cardiac events. <Example 5> Blood PTX 3 concentration and patient clinical background (1)>

 Blood was collected at the time of enrollment in the study and subjected to biochemical tests such as plasma ΡΤΧ3, high-sensitivity CRP (C-reactive protein), and ΒΝ Ρ (brain sodium peptide). Two-dimensional echocardiography was also performed by a cardiologist within one week of registration. The doctor in charge of the treatment was not informed of the results of the biochemical examination, and the optimal medical treatment was performed based on symptomatic improvement, physiologic examination findings, and pulmonary congestion findings from chest X-ray examination. Table 1 shows the clinical background of the heart failure patient group.

 The cut-off value of plasma ΡΤΧ3 concentration was 4.4 ng / mL and divided into 2 groups. Table 1 shows the results of comparing clinical backgrounds of the two groups. To exclude patients with factors affecting PTX3, patients with renal insufficiency with serum creatinine levels higher than 2. Omg / d L, patients with systemic inflammatory disease or collagen disease in active phase Patients with acute coronary syndromes clinically or electrocardiographically suggested within 3 months before enrollment were excluded (n = 1 34, hereafter referred to as exclusion group).

[0070]

1

PTX3 normal value group PTX3 high value group

 (<4.4 ng) (n = 68) 04.4 ng / mL) (n = 66) P value Age (age) 68.5 Sat 10.6 67.9 Sat 15.5 0.8071 Gender (Male 1 Female) 41 127 35 131 0.3967 Heart function classification (I / II / III / IV) 21/31/15/16/18/32/10 0.0001 Hypertension 34 (50%) 26 (39¾) 0.2171 Diabetes 17 (25¾) 14 (21¾) 0.6032 Hyperlipidemia 12 (18¾) 11 (17¾) 0.8804 Etiology of chronic heart failure 0.5056

 Dilated cardiomyopathy 20 (29%) 19 (29)

 Ischemic heart disease 19 (28%) 17 (26%)

 Valvular heart disease 11 (16%) 12 (18¾)

 Hypertensive heart disease 10 (15%) 7 (11¾)

 Other 8 (12¾) 11 (16¾)

 ■ Echocardiography

 Left end diastole diameter (mm) 53.3 Sat 9.7 54.9 Sat 11.3 0.4188 Left ventricular ejection fraction is 51.7 + 19.4 44 4 Sat 18.0 0.0267

BNP (pg / iL) 377.9 Sat 534.1 825.8 + 739.9 + 0.0001 Serum creatinine (mg / dL> 0.87 Sat 0.29 0.93 Sat 0.31 0.3121 Uric acid (mg / dL) 6.1 Sat 1.8 6.5 ± 2.3 0.2955

Na (mEa / L) 141.3 1 ± 2.8 140.5 ± 3.3 0.1030 High sensitivity CRP (mg / dL) 0.30 + 0.67 1.13 Sat 1.31 + 0.0001 Notes: YH New York Heart Association: BNP, brain natriuretic peptide; CRP, C-reactive protein PTX3, Pentraxin 3; ACE, angiotensin converting enzyme inhibitor; ARB, angiotensin II receptor protocoler <Example 6 Blood PTX 3 concentration and occurrence of cardiac event (1)>

There were 42 cardiaceents (9 deaths, 33 readmissions) during the observation period of the exclusion group (mean 546 ± 327 days, range 5–11 77 days). Here, cardiac event means death or readmission due to worsening heart failure. Divided into 2 groups with and without cardiac event, S Table 2 shows the results of a clinical background comparison using tat View Ver. 5.0 (SAS Institute Institute USA).

 2]

Without Cardiac event With Cardiac event

 (n = 92) (η = 42) 年 齢 Age (age) 67.1 ± 13.5 70.7 ± 12.4 0.1448 Gender (male / female) 51/41 25117 0.6576

NYHA Cardiac Classification (I 1 II / III / IV) 26/37/23/61/12/24/5 <0.0001 Hypertension 44 (48¾) 16 (33¾) 0.2933 Diabetes 21 (23¾) 10 (24%) 0.9003 High Lipemia 20 (22¾) 3 (7¾) 0.1681 Etiology of chronic heart failure 0.4424 Dilated cardiomyopathy 24 (26¾) 15 (36¾)

 Ischemic heart disease 26 (28¾) 10 (24¾)

 Valvular heart disease 16 (18¾) 7 (则

 Hypertensive heart disease 13 (14%) 4 (则

 Other 13 Congress 6 (14¾) Echocardiography

 Left end diastolic diameter (mm) 52.7 + 9.4 57.5 Sat 12.1 0.0169 Left ventricular ejection fraction is 51.1 ± 19.1 41.2 ± 17.1 0.0056

BNP (pg / niL) 446.9 ± 647.5930.7 Sat 634.5 + 0.0001 Serum creatinine (mg / dL) 0.84 Sat 0.26 1.04 Sat 0.32 0.0002 Uric acid (mg / dL> 6 ± 1 ± 2.1 6.7 ± 1.9 0.0960

Na (mEa / L) 141.2 ± 2.5 140.3 ± 3.9 0. 112 High sensitivity CRP (mg / dL) 0.64 soil 1.04 0.86 soil 1.28 0.2856

PTX3 (ng / niL) 5.3 Sat 5.5 15.2 Sat 26.8 0.0027 Note: Same as Table 1. Death or readmission due to worsening heart failure in the non-excluded group is defined as Cardiac E vnt, with an average endpoint of 546 days (546 ± 327 days Range 5 ~ 1 1 77 曰) Follow-up. S tat V iew V er. 5.0, (SAS institute I n c. U SA) was used to create a survival curve using the Kaplan-Meier method, and the cumulative cardiac between the two groups divided by plasma PTX 3 concentration. As a result of log rank test of the percentage of patients who passed without E vent, p = 0. 0003 (PTX 3 high value group 53.0% v s. Ding 3 low value group 83.8%) (Figure 5). ) The percentage without Cardiac E vent was significantly lower in the high PTX 3 group than in the low PTX 3 group. Results of a univariate Co X proportional hazards analysis (SAS institute I n c. USA) to assess plasma PTX 3 concentrations as a predictor of the occurrence of cardiac vent Is shown in Table 3. The increase in plasma PTX 3 concentration by 1 SD (16.1 ng / mL) was a significant variable (hazard ratio 1.23, 95% confidence interval 1.05—1.44, p = 0. 0 1 25).

3) Variable Hazard Ratio 95 Confidence Zone P Value Gender (Every 5 years old) 1.03 0.99-1.05 0.0666 Male VS Female 1.12 0.60-2.07 0.7240

NYHA classification I / II YS III / IY 3.94 2.03-7.63 <0.0001 Presence of disease

 Hypertension 0.67 0.36-1.24 0.2023 Urinary disease 0.98 0.48-2.00 0.9611 Hyperlipidemia 1.77 0.87-3.60 0.1166 Echocardiography

 Left ventricular end-diastolic diameter (every 1SD increase) 1.60 1.23-4.09 0.0076 Left ventricular ejection fraction (every 1SD increase) 0.59 0.42-0.83 0.0020

BNPdSD increase) 1.98 1.00-1.98 0.0003 Creatinine (1SD increase) 1.76 1.36-2.29 <0.0001 Uric acid (1SD increase) 1.39 1.04-1.85 0.0265

Na (every lSD increase) 0.79 0.58-1.06 0.1238

 High sensitivity CRP (Every 1SD increase) 1.11 0.85-1.46 0.4472

PTX3 (Every 1SD increase) 1.23 1.05-1.44 0.0125 Presence or absence of medication

 ACE inhibitor or MB 1.39 0.70-2.78 0.3496 j8 blocker 1.22 0.66-2.26 0.5288

Ca channel blocker 0.58 0.24-1.38 0.2182 Diuretic 6.46 2.30-18.15 0.0004 Subtilin 1.45 0.60-3.52 0.4158 Note: Same as Table 1

[0075] Furthermore, Table 4 shows the results of multivariate Co X proportional hazard regression analysis using variables whose p-value was less than 0.05 in the results of Table 3. 1 SD change in plasma PTX 3 concentration was the only independent and significant factor for predicting the occurrence of cardiac vent (hazard ratio 1.29, 95% confidence interval 1.07— 1. 58, p = 0.0074).

[0076] 4) Hazard ratio 95% confidence zone Left ventricular end-diastolic diameter (every 1SD increase) 1.35 0.78-3.55 0.1840 Left ventricular ejection fraction (every 1SD increase) 0.93 0.57-1.51 0.7575

BNP (Every 1SD increase) 1.25 1.00-1.98 0.1228 Uric acid (Every 1SD increase) 1.19 0.81-1.76 0.3663 Creatinine (Every 1SD increase) 1.41 1.00-2.00 0.0526

PTX3 (Every 1SD increase) 1.29 1.07-1.58 0.0074 Note: Same as Table 1

 [0077] Next, FIG. 6 shows the results of a similar quartile analysis of 144 non-exclusion groups. Relative risk was highest in the fourth quartile group (6.85 times that in the first quartile group). 1st quartile group (<2.5 ng / mL), 2nd quartile group (2.5-4. 3 ng / mL), 3rd quartile group (4.3-3-7. 0 ng L) and the fourth quartile group (> 7.0 ng / mL), p = 0.0482, the first quartile group between the first and third quartile groups. Between the rank group and the fourth quartile group, p = 0. 0053,

 P

 Values There was a significant difference between the second and fourth quartile groups at p = 0.0073 (Figure 6).

 <Example 7> Blood PTX3 concentration and patient clinical background (2)>

 We examined the clinical significance of prognostic factors for chronic heart failure (CH F) in the determination of PTX3 concentration in patients with heart failure who visited Saga University School of Medicine. P TX 3 was measured by the ELISA method described in Example 1. 37 CHF patients were divided into two groups according to plasma PTX3 concentration (3.06 ng / mL), and the main clinical background items were compared. Not recognized (Table 5).

[0079] 5

Cn = 19) (n = 18) Male tfc¾li 11/6 10/10 0.37 Age is> 63 + 14 70 ± 10 0.07

NYHA * B (πθΰ 6C32) 7 ¾5> 0.99

 3 (16) 7 C35) 0.24 Sugar ^ ϊη <*> 4 (21) 5 (25) Q92

 59 ± 10 59 ± 5 087

34 ± S 30 ± 9 0.18

1509 13 (65) 0.10 -Blocker 16 <&) 01

 15 J9) 13 (65) 0.10 Digitalis 7C37) 4 (20) ai6 Suwon 5 (25)

 269 ± 202 311 ± 324 Q64 hsCRP, mfiA _ ++++ 6.3 ± 9.2 2.4 ± 6_0 ai3 York Heart Association heart function ¾iS

 ** ACEI / ARB: Angiotensin post-change II element or angiotensin receptor blocker

 Ft ft na Wum diuretic betide

 ** ++ l Reactive tongue J S KU G¾¾Jg)

[0080] <Example 8 Generation of blood PTX3 concentration and cardioacevent (2)>

 During the observation period of 30 months (average: 10 ± 6 months, range: 1 to 23 months), a comparison was made between the group in which an event occurred (event group) and the group in which no event occurred (no event group). (Table 6). Here, event occurrence means death or readmission due to worsening heart failure. As a result, significant differences were observed in NYHA classification, serum CRP concentration, and plasma PTX3 concentration.

[0081]

6)

 21/18 4/2 17/14

 Year (age) 66 ± t3 67.7 + 13.1 63.7 ± 13.0

ΥΗΑ * Έ Ν _Λ η <> 17 <46> 5 (83) 0.007

 10 <≥3> 2C0) 8¾δ> n

8 (23> 2) 6≥1> n &

¾E¾¾ ^ B I, 61 + 14 59 + 7 as.

 * 32 ± 9 35 ± 6 31 ± 9 a bride πΟΟ

 2604> 4 (67) 22 C76) na j3-blocker 32 <91> 583) 28 (97)

 髓 26 (74) 4 <67) 22 G6) ns. Di =? Ris 11 <31) 3 <K »8 ^ 8)

 (10 <≥9> 2 33)

 BN stsf ** 285 + 276 362 ± 301 2? 0 ± 274

 hGC R, me L **** 4.4 + 7.8 9-2 + 10.7 3.4 ± 7.1 UGQ

PTX3 ng / ml 4.1 ± 25 7.0 ± 2 ^ 3.5 ± 2.8 αοοθ

* Comments are the same as in Table 5. In addition, 37 CHF patients were arranged in order of increasing concentration using Ρ Τ X 3 concentration as a variable, separated by equal percentiles and divided into 3 groups (low value group: <2.4 ng / mL, medium Value group: 2.4-4. 2 ng / ml, High value group:> 4.2 ng / ml) Observational studies were conducted. The average plasma PTX3 concentration in all CH F patients was 4.1 ± 2.9 ng / mL, which was higher than the average value in the healthy control group 2.1 ± 0.9 ng / mL (p = 0 001) 1 During the 0-month observation period, 1 patient died due to CH F exacerbation and 5 patients were readmitted due to CH F exacerbation. As a result of a 30-month follow-up and log rank test, significantly more events were observed in the plasma high PTX 3 group than in the low PTX 3 group (42% v s. 0% p = 0. 02) (Figure 7). In addition, Cox multivariate regression analysis (SAS institute Ic. USA) was used to determine age, gender, NYHA heart function classification class, left ventricular ejection fraction, high-sensitivity serum CRP (C-reactive protein) concentration, BN Analysis was performed using P (brain sodium peptide) and PTX3 as variables. As a result, a significant difference was observed from the high-sensitivity serum CRP concentration, which is currently regarded as a prognostic factor for CHF (Table 7). This allows PTX 3 is a predictor of the occurrence of events such as significant death or rehospitalization, and can be predicted to hate CH F patients (hazard ratio 1.91 2, p = 0.019, 95% confidence Section 1. 1 1 4— 3. 282).

[0083] [Table 7] Hazard ito

 Textile 纖 Hazard ratio 95Κ ^ Μΐ; Between men 1.488 9.167 0.498-169.338 0.138

 Q047 0.997 0 Fine-1.093 0.947

ΝΥΗΑΠΙIV 1.948 90. & 46 1 腦 ^ 136.87? 0.021 Miki 0.120 1.272 1.006-1 βθθ ao

ΒΝΡ 0.0Q2 1.0Q2 997- 1.007 tt399 bsCRP Q083 0942 0.801-1.107

 PTX3 0576 1- "4-3.282 0.019

[0084] The data on the production of the anti-PTX 3 monoclonal antibody used in the ELISA method shown in Example 1 and the performance of the ELISA method measurement kit are shown below.

 <Example 9 Cloning of Ding 3>

Cloning of the sequence containing the full length ORF region of PTX 3 was performed. Single-strand cDNA of human umbilical vein endothelial cells (HUVEC) is synthesized from mRNA using reverse transcriptase. cDNA synthesis is performed using AMV Reverse Transcr Iptase First—strandc DNA Synthesis Kit (manufactured by Seikagaku Corporation). For cDNA synthesis and amplification, 5'-Amp I i FIN DER RACE Kit (CI ontech) and PC's 5'-RACE method (Frohman, MA eta I. , Proc. N at I. A ca d. S ci. USA (1 988) 85, 8998—9002, Be I yavsky, A. eta, Nucleic A cids Res. (1 989) 1 7 , 291 9-2932) etc. can be used. Using it as a cage, primers _ PTX 3-F (K pn I) (SEQ ID NO: 03) and PTX3_R (BamH I) (SEQ ID NO: 04) designed from G en Bank number (NM—002852) The full-length ORF gene was isolated by PCR. . The fragment obtained by PCR was inserted into a vector using Zero Blunt TOPO PCR Cloning Kit, and the base sequence analysis was carried out by a conventional method. Then, it was applied to K pn I site and B am H I site. The fragment thus cut was inserted into a ph CMV vector (Stratagene) to prepare a transfer vector ph CMV-PTX3.

 <Example 10 Construction of 0-full-length PTX3-expressing cells>

According to the protocol of Fu GEN E 6 (Ro che Molecular Bio Biotechnology), seeded 1 X 10 ⁵ cell C HO cells on 6 days before transfer The next day, 8 g of expression vector ph CMV_PTX3 and 16 L of FuGEN E 6 reagent were mixed with 100 L of serum-free DMEM medium and incubated at room temperature for 20 minutes. Added to cells. The next day after transfection, cloning was performed using the limiting dilution method and G 418, which is a selective reagent. The culture supernatant of each clone was collected and screened for PTX 3 protein expression cells. As a result, it was possible to select a clone (hereinafter referred to as CHO-PTX3) that constantly expresses about 2 to 3 g / mL of PTX3 protein.

 <Example 1 1 Obtaining Recombinant PTX 3 Protein>

Protein purification was carried out according to the method of Bottazzi et al. (Bottazzi B, Vouret—Caviari, eta I., JB iol Chem. 1 997; 272 (52): 3281 7-23.). Specifically, after cultivating CHO-PTX3 in a flask of 150 cm ² , using a roller one pot (BD Bioscience), 30 OmL serum-free medium (S-S FM-II, GI BCO / Invitrogen), the rolling speed was adjusted to 1 rotation per minute, and the culture was cultured for 4 days, and the culture supernatant was collected. 1 L of the culture supernatant was concentrated to 5 Oml using an ultrafiltration membrane concentrator, pel I icon XL device Biomax 100 (MILLIPO RE). Concentrate 50 mM I mid Dialyzed against 5 L of azo Ie, pH 6.6 buffer. Dialysis was performed twice against the same buffer. Subsequently, it was applied to ion exchange chromatography using HiPrep 16/10 QXL (Pharmacia Biotech, Uppsala, Sweden). After washing to a low background level, the concentration of NaCI was increased from 0 to 0.58M over 35 minutes, and then PTX3 was eluted with 1M NaCI. The elution was monitored at an absorbance of 280 nm. Fractions containing PTX 3 protein were collected, and gel filtration chromatography using Sephacry IS_300 was developed on PBS. Furthermore, in order to obtain only multimeric PTX 3 out of PTX 3, purification was performed using Superose 6 col umn (GE Healthcare). That is, after carrying out a calibration using a molecular weight standard, PTX 3 was applied to the column and eluted with PBS at a flow rate of 0.4 mL / min. Each elution fraction was analyzed by SDS-PAGE to obtain a purified recombinant multimeric PTX 3 protein. <Example 1 2 Identity of Recombinant PTX3 Protein and PTX3 Molecular Weight in Clinical Specimen>

 Example 1 The molecular weight of the recombinant PTX 3 protein obtained in 1 and the PT X 3 protein in clinical samples was obtained by gel filtration using Superose 6 col umn (G 巳 Healthcare). And analyzed. In other words, after calibrating using molecular weight standards, a sample in which recombinant PTX 3 protein was added to human plasma with a PTX 3 concentration below the measurement limit to 30 ng / mL was applied to the column. Elution was performed with a buffer solution (20 mM HEP ES, 15 mM NaCI, 0.05% sodium azide, pH 7.2) at a flow rate of 0.3 mL / min. Gel filtration was performed under the same conditions using a clinical specimen (plasma) in which PTX3 concentration was detected at 10 ng / ml by the ELISA method. The PTX3 concentration of each elution fraction was detected by the ELISA method.

As a result, the recombinant PTX 3 protein and PTX 3 in clinical samples A peak was observed in the elution fraction of about 900 kDa from the calibration using the molecular weight standard. (Figure 8)

<Example 1 3 Production of anti-PTX3 monoclonal antibody>

 A monoclonal antibody was prepared by the following procedure. That is, Ba Ib / C mice (CRL) or PTX 3 knockout mice were immunized with PTX3. For the first immunization, the immune protein was prepared at 100 g / animal and FCA (Freund's complete adjuvant (H 37 Ra), D ifco (31 1 3-60), Becton Dickinson (cat # 231 1 31 )) Was emulsified and administered subcutaneously. Two weeks later, 50 g; U g / animal prepared with FIA (Freund's incomplete adjuvant, D ifco (0639-60), Becton Dickinson (cat # 26391 0)) It was administered subcutaneously. Thereafter, booster immunization was performed twice at a weekly interval. The final immunization was diluted in PBS to give 50 U animals and administered into the tail vein.

 After confirming that the antibody titer in serum against PTX 3 was saturated by ELISA using an immunoplate coated with PTX 3 protein, mixed mouse myeloma cells P 3 U 1 and mouse spleen cells. Cell fusion was performed with P EG 1 500 (Roche-Diagnostic, cat # 783 641). After seeding in a 96-well culture plate, the culture supernatant was screened with ELISA as follows after selection with HAT medium from the next day. The full-length PTX 3 described in Example 11 was immobilized on a solid phase, and after incubation with the culture supernatant of a hybridoma, the antigen solid phase E L ISA method in which detection was performed with a labeled anti-mouse antibody was performed.

 Positive clones were monocloned by the limiting dilution method and then cultured by expansion culture, and the supernatant was collected. Screening with ELISA was performed using the binding activity with PTX 3 protein as an index, and many anti-PTX 3 antibodies having strong binding ability were obtained.

[0090] Monoclonal antibody was purified using H i Trap Protein GHP (G E Healthcare). The supernatant of the hybridoma culture was directly charged onto the column, washed with binding buffer (20 mM sodium phosphate (pH 7.0)), and then eluted with elution buffer (0.1 M glycine—HC I (pH 2. 7) Dissolved in). The eluate was collected in a tube containing neutralization buffer (1 MT ris -HCI (pH 9.0)) and immediately neutralized. The antibody-eluted fractions were pooled, and dialyzed overnight with 0.05% Tween 20 / PBS to replace the buffer solution. Na N ₃ was added so that the purified antibody was 0.02%, and then stored at 4 ° C.

 <Example 1 4 Subclass of 4 Anti-P T X 3 Monoclonal Antibody>

 The anti-PTX3 antibody was isotyped using ImmunoPureMonoclnaal lAntiboddyIsotyPingKitII (PIERCE CAT # 37502), and the method was performed according to the attached manual. As a result, a large number of IgG1, IgG2a, and IgM class antibodies were obtained. P PMX 1 04 and P PMX 1 05 were both I g G 1

<Example 1> Preparation of polypeptide (N_PTX3) of N-terminal portion of 5PTX3>

 The polypeptide at the N-terminal part of PTX3 (N—PTX3) was expressed in E. coli as a GST (glutathione_S_transferase) fusion protein and purified. The expression vector for N-PTX 3 was constructed using a general method. That is, the expression vector of N-PTX3 was amplified by the PCR method using the full-length PTX3 as a template and nucleotide sequences 1 to 522 using appropriate primers of SEQ ID NOs: 5 and 6. Next, the p ENTR TM / D—TOPO Cloning Kit Gat ewa y (R) System and p DES TTM24 Vector I (both from Invitrogen) were used to construct a vector according to the usage capacity of the attached manual. It was.

Transform the constructed vector into E. coli BL 21 S tar TM (DE3) and induce expression of the target gene by inducing expression with arabinose. went. Next, the expression-induced E. coli cells are recovered, solubilized in a buffer containing a surfactant such as NP40, and lysozyme. After centrifugation, the supernatant is recovered and purified using a GST column. It was. That is, the fusion protein was bound to GST cephalose and beads were washed with PBS. Thereafter, the fusion protein was eluted with a reduced glutathione solution. The purified protein was subjected to SDS_PAGE electrophoresis and used as an antigen for antigen solid phase ELISA after purity, molecular weight and other tests.

<Example 1 6 Epitope analysis using polypeptide (N- PTX3) of N-terminal part of PTX3>

 The binding site of the anti-PTX3 monoclonal antibody (P PMX01 01, P PMX01 02, P PMX01 04, P PMX01 05) to the PTX3 protein was identified as follows.

The full-length PTX 3 protein obtained in Example 11 and the polypeptide of the N-terminal part of PTX 3 described in Example 15 (N—PTX3) were used as materials. As a method for specifying the binding site, a general antigen solid-phase ELISA method was used. That is, these proteins were prepared to 5 g / mL, 100 L / we II was added to the ELI SA pre-coagulum, and solid-phased by a 4 ° C-coagulation reaction. The next day, after washing 3 times with 300; UL / we II wash buffer (0.05% (v / v) Tween 20, PBS), TBS containing 10% Block Ace (Dainippon Pharmaceutical) (10 mM) Blocking was performed by adding 1 5 of Tris-HCI, 150 mM NaCI, pH 7.5). After several hours at room temperature or after storage at 4 ° C for a while, culture supernatant of diluted hybridoma containing monoclonal antibody or diluted purified monoclonal antibody was added with 1 OOL / we II and incubated for 2 hours at room temperature. Next, peroxidase-labeled anti-mouse I diluted 5000 times with TBS (1 OmM Tris-HCI, 150 mM MN a CI, pH 7.5) containing 10% Block Ace (Dainippon Pharmaceutical) g Gagi I gG (Cappel) was added at 100 L / we II and incubated at room temperature for 2 hours. 300; UL / we l I wash buffer After washing 5 times with —, color was developed using Scytek TMB (Cat # TM49 9 9) according to the attached protocol, and the absorbance was measured with a microplate reader.

 The recognition site was identified for a monoclonal antibody that showed high absorbance with the full-length PTX3 protein.

 [0094] <Example 1 7 Epitope analysis by Western plotting using full length PTX3 protein>

 Example 11 The full-length recombinant PTX3 purified in 1 was treated with a sample buffer under reducing and non-reducing conditions, 60 ng was applied per lane, and SDS-PAGE was performed. Next, the membrane was transferred to a Hybond_ECL (GE Healthcare) membrane at 38 V for 16 hours, and the protein was transferred to the membrane, followed by blocking with Block Ace (snow mark) for 1 hour at room temperature. Next, 0.3 g of anti-PTX 3 antibody was added to 40% Block Ace (Snow Mark) / TBS solution, reacted at room temperature for 1 hour, TBST (50 mM Tris— HCI (pH 7.5), Washing was performed 5 times x 3 times with 1 5 OmM NaCI, 0.05% Tween 20). Then, add HRP-labeled anti-mouse IgG antibody (GE Healthcare) diluted 10000 times with 10% Block Ace (Snow) / TBS, and let it react at room temperature for 1 hour Washed 3 times with TBST. Finally, an ECL detection reagent (GE Healthcare) was allowed to act, and the resulting chemiluminescence signal was exposed to an X-ray film for 5 minutes.

 Under reducing conditions, P PMX 0 1 0 1 and P PMX 0 1 02 reacted with full-length P T X 3, and P PMX 0 1 04 and P PMX 0 1 05 showed no reactivity. Under non-reducing conditions, both antibodies reacted with full-length PTX3 (Figure 9).

 [0095] The hybridoma of P PMX 0 1 02, which produces a monoclonal antibody that recognizes PTX 3, is FE RM BP— 1 03 2 6. It was deposited in Tsukuba City East 1 1 1 1 1 Chuo No. 6) (Deposit Date: February 15, 2005).

<Example 1> 8 Preparation of PTX 3 C-terminal Polypeptide (C_PTX 3) >

 Forced expression of the polypeptide at the C-terminal part of PTX3 (C_PTX3) (positions 1 79 to 381 of SEQ ID NO: 2) in a CHO cell line was performed by the following method. In addition, the N-terminal polypeptide (N—) encoding the amino acid at positions 1 to 151 of SEQ ID NO: 2 is different from the N-terminal polypeptide (N—PTX3) prepared in Example 16. PTX3 (2)) was also prepared.

 First, the human PTX 3 cloned in Example 9 was made into a saddle type, and cDNA encoding the amino acids 179 to 381 of PTX3 was amplified by PCR, and pSG5 vector 1 (stratagene) An expression vector was constructed at the BamHI site. c When obtaining DNA, a primer was synthesized and PCR amplification was performed so that the PTX3 signal peptide was added to the 3 'side.

Preparation of PTX 3 partial length protein forced expression in CHO cells was carried out by the following procedure. 1 0 cm dish O. 8 x 1 0 ⁶ CHO cells are seeded and the next day! "8 g of plasmid DNA was transfected using u GEN t 6 1 ransfection Reagent (Ro che).

 After 48 hours, the cells were detached and collected with a cell scraper. 200 1_ I PA buffer (10 mM Tris_CI, 150 mM NaCI, 5 mM EDTA, 1% Triton X_1 00, 1% deoxycholate 0.1% SDS, pH 7.4) was added, and the cells were lysed by placing on ice for 15 minutes. Subsequently, centrifugation was performed at 15,000 xg, 15 minutes, 4 ° C, and the supernatant was used as an expressed protein solution.

Different from the N-terminal polypeptide prepared in Example 16 (N_PTX 3), the N-terminal polypeptide (N- PTX3) encoding the amino acid at positions 1 to 1 of SEQ ID NO: 2 In (2)), a PTX 3 partial length polypeptide at positions 1-151 was obtained in the same manner as for C-PTX3. However, since the N-terminal has a signal polypeptide at position 1 _ 17, no signal polypeptide is added. Absent.

 Next, the expression product was confirmed. Treat expressed protein samples with sample buffer under reducing conditions (2—ME added) or non-reducing conditions (2 _M E added), apply 20 g of expressed protein solution per lane, SDS-polyacrylamide gel electrophoresis After transfer to Hybond_P (GE Healthcare), the membrane was soaked in 100% Block Ace (Snow Brand Milk Products) and shaken at room temperature for 1 hour for blocking. Next, the membrane was immersed in a suspension of serum collected from a mouse sensitized with full-length PTX 3 in 40% Block Ace / TBS (1 OmM Tris_Cl, 150 mM NaCI, pH 7.5). The primary reaction was performed by shaking at room temperature for 1 hour.

 After washing with TBST (TBS + 0.1% Tween 20) for 5 min x 2 times, block HRP-labeled anti-mouse Ig G (GE Healthcare, cat. NA 9 3 1) with 10% The membrane was immersed in a solution diluted 5000 times with ace / TBS, and shaken at room temperature for 1 hour to carry out a secondary reaction. After washing with T B S T (T B S +0.1% T ween 20) for 5 minutes × 3 times, coloring was performed using E C L (GE Healthcare) (FIG. 10). In addition, as a negative control, the culture supernatant of Hyb-342 3 that produces a monoclonal antibody against hepatitis B virus S antigen (HBs antigen) that does not recognize PTX 3 as the primary antibody, and the secondary antibody as anti-mouse Ig Western blotting was performed as a G antibody (GE Healthcare) (Fig. 11).

<Example 1 9 Epitope analysis using polypeptide (C_PTX3) at the C-terminal part of PTX3>

Example 1 CHO cell lysate expressing N-terminal (N-PTX 3 (2)) and C-terminal polypeptide (C-PTX 3) prepared in Example 8, lysis of CHO cells without gene introduction Liquid, purified full-length recombinant PTX 3 was treated with a sample / buffer under the conditions of reduction (2_ME added (+ 2—ME)) and non-reduced (2—ME not added (_2_ME)). Apply 20 μg of cell lysate per lane, 3 ng of full-length PTX 3 and use P PMX O 104 as the primary antibody. Example 17 A stamp stamp lot was made by the method described in 7.

[0098] As a result, P PMX 01 04 was reduced to full length PTX 3, N-terminal polypeptide (N—PTX3 (2)), and C-terminal polypeptide (C—PTX 3) under reducing conditions. I did not recognize. In non-reducing conditions, it reacted only with full-length PTX3 (Fig. 12).

[Example 20] Reactivity of protease degradation product of full-length PTX 3 protein and monoclonal antibody under reducing conditions>

 In order to identify the binding site to PTX3 protein in more detail, PTX3 was fragmented by enzymatic digestion, and the fragments were fractionated by reverse phase HPLC. Thereafter, the reactivity to the collected peptides was examined by the ELISA method.

 First, the Ding 3 protein was dissolved in 0.5M Tris—HC I, 6 M guanidine—HC I, 10 mM EDTA, pH 8.5, and 31.5 times the DTT of PTX 3 in molar ratio. In addition, it was left at 37 ° C for 2 hours for reduction treatment. Next, 4-vinyl monopyridine, which is 3.1 times the amount of DTT in terms of molar ratio, was added, and the mixture was allowed to stand at room temperature for 2 hours in the dark to perform pyridylethylation of the SH group. Next, this was dialyzed against pure water and then 50 mM Tris—HC I, 3M urea, pH 9.0, so that the molar ratio was 1/50 of PTX 3 and Lysyl Endopeptidase (Wako Pure Chemical Industries) was added, 37. C, 18 hours reaction was performed, and PTX 3 protein was cleaved. Enzyme digest was applied to a Symme try 300 C 18 column (Waters) and eluted with 0 to 60% gradient of acetonitrile for 1 minute (flow rate 0.8 mL / min). (Figure 1 3).

These fragments were diluted 50-fold with PBS, 100 L / we II was added to the ELISA plate, and solid-phased by a 4 ° C-IV reaction. The ELI SA method was performed as follows. After immobilization, wash 3 times with 300; UL / we II wash buffer-(0. 05% (v / v) Tween 20, PBS) and then 40% Block Ace (Dainippon Pharmaceutical). Add 1 50 1_ of TBS (1 OmM Tris_H CI, 1 5 OmM NaCI, pH 7.5) Went. After several hours at room temperature or after storage at 4 ° C, the P PMX 0 10 4 or P PMX O 1 05 antibody is dissolved in the same solution as the blocking solution to a final concentration of 10 U g / m I 100 L / we II was added and incubated for 2 hours at room temperature.

 Next, peroxidase-labeled anti-antibodies diluted 5,000 times with TBS (10 mM MT ris-HCI, 150 mM NaCI, pH 7.5) containing 10% Block Ace (Dainippon Pharmaceutical). Mouse Ig Gagi IgG (Cappe I) was added at 100; UL / we II and incubated at room temperature for 2 hours. 3 After washing 5 times with 00 UL / we II wash buffer, develop color using Cytek's TMB (Cat # TM49 9 9) according to the attached protocol and measure absorbance with a microplate reader. It was measured.

 As a result, it did not react at all with the peptide after enzymatic cleavage (Figs. 14 and 15). From this fact, it was estimated that P PMX O 104 and P PMX O 105 were antibodies that recognize the three-dimensional structure of PTX3. In addition, P PMX 0 04 and P PMX 0 1 05 are composed of S—S bonds because they have almost lost reactivity with PTX 3 before enzymatic cleavage, that is, reduced pyridylylated. It can be seen that this antibody recognizes the three-dimensional structure of PTX 3.

<Example 21 Reactivity of lysyl peptidase degradation product of full length PTX3 protein and monoclonal antibody under non-reducing conditions>

 Example 1 The full-length PTX 3 protein obtained in 1 was added to 0, 0.5, 1, 2, 4, 8 in 200 mM Tris-HCl buffer using lysylendopeptidase (lysylendopeptidase). Set the digestion time and digest at 30 ° C. After digestion time, digestion is stopped by adding DFP (Fluorophosphate Disopropyl), the digested sample is fractionated by SDS_PAGE analysis, the gel is stained with Kumashi Brilliant Blue (CBB), and PTX 3 is cleaved (Fig. 16).

Conventional solid-phase ILISA method using lysylated peptidase digested sample The reactivity with anti-PTX 3 monoclonal antibody Ρ ΡΜΧ01 04, Ρ ΡΜΧ01 0 5 was confirmed. Specifically, the lysyl endopeptidease digested sample was immobilized on an ELIS plate and reacted with P PMX01 04 or P PMX01 05 as the primary antibody, and horseradish peroxidase labeled anti-mouse. ELISA was performed using Ig antibody antibody (GE Healthcare) as a secondary antibody. The results of ELISA are shown in Table 1 as absorbance values. Figures 17 and 18 show the relationship between the lysyl-peptidase digestion time of PTX3 and the ELISA results.

 [0101] From these results, the hybridomas of P PMX 01 04 and P PMX 01 05 that produce monoclonal antibodies that recognize the three-dimensional structure of PTX 3 are FERM BP— 1 07 1 9 and FE RM BP, respectively. — 1 0720 Deposited at National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Address: East 1st, Tsukuba City, Ibaraki Pref. 6th Central) (FE RM BP— 1 07 1 9 and FE RM B P- 1 0720 : Date of deposit: September 22, 2005).

 <Example 22: Measurement of dissociation constant of PTX 3 monoclonal antibody>

 The coupling constant was measured using a BIAcor 3000 system (BIAcorre, Uppsala, Sweden). First, an anti-mouse Ig G antibody was immobilized on the sensor chip CM5 using the NHSS / EDC coupling method. Next, anti-PTX 3 antibodies (P PMX01 04, P PMX 01 05) were added to HBS—EP buffer (10 mM H EP ES, pH 7.4, 150 mM NaCI, 3 mM EDTA, 0.005% A suspension of 10 μg / mL in surfactant P 20) was injected to immobilize an antibody of several hundred RU. Next, a suspension of the recombinant PTX3 in HBS—EP buffer was measured, and the binding and dissociation were measured. Then, the dissociation constant was obtained using an analysis program (BIAeva luation).

[0103] As a result, both P PMX 01 04 and P PMX 01 05 produced this time showed low dissociation constants. Among them, P PMX01 04 had the lowest dissociation constant. And showed high affinity. (Table 8).

[0104] [Table 8] Dissociation constants of anti-ΠΧ3 antibody

<Example 2 3 Cross-reactivity of anti-PTX3 monoclonal antibody with SAP and CRP>

 Confirmation of cross-reactivity of P PMX 0 1 04 and P PMX 0 1 05 with S A P and C R P was performed by a general antigen-immobilized E L I S A method. That is, prepare full length PTX 3 protein, human CRP (Japan Biotest Laboratories), human SAP (Wako Pure Chemical Industries) to 5 g / mL, add 100 UL / we II to the ELISA plate, Solid phase immobilization was carried out by a reaction of 4 ° C-IV. The next day, 300 μL / we II was washed; 'Conflict / Kuffaichi (0.05% (v / v) Tween 20, PBS) was washed 3 times and contained 40% Block Ace (Dainippon Pharmaceutical). TBS (10 mM Tris-HCI, 150 mM NaCI, pH 7.5) was added and blocked. After several hours at room temperature or after storage at 4 ° C, add the culture supernatant of the hybridoma containing the monoclonal antibody or diluted purified monoclonal antibody at 100 L / we II and incubate for 2 hours at room temperature. did. Next, peroxidase labeling diluted 5,000 times with TBS (10 mM Tris-HCI, 15 OmM NaCI, pH 7.5) containing 10% Block Ace (Dainippon Pharmaceutical) Anti-mouse IgG goat IgG (Cappel) was added at 100 L / we II and incubated at room temperature for 2 hours. 300; Wash 5 times with a wash buffer of UL / wel I, develop color using Scytek TMB (Cat # TM49 9 9) according to the attached protocol, and measure the absorbance with a microplate reader. It was measured.

Monoclonal antibody P PMX 0 1 04 and P PMX 0 1 05 Reacts strongly to TX 3, but not to SA CR or CR 全 く (Table

9).

[0106] [Table 9]

Anti-PPMX0104 and PPMX0105 against PTX3, CRP and SAP

 Responsive

<Example 24: F (a b ') conversion of antibody>

F (ab ') 2 conversion of the antibody was performed as follows. The antibody purified by the method for producing a monoclonal antibody described in Example 4 was dialyzed against a dilute buffer (5 mM Tris—HCI, 15 OmM NaCI, pH 7.5). For the antibodies of subclass I g G 1, use pH 3.7 for pH 3 and for antibodies of Ig G 2 a pH 4.0 digestion buffer (0.2 M Sodium citrate buffer). Dilute 2 times, 37. Incubated at C for 5 minutes. Next, prepare 1 mg ₈ ml of pepsin solution in P epsin digestion buffer of ρ Η4 · 0 and add pepsin solution to the heated sample. Was added at a mass ratio of 1: 30: 1 (antibody: Pepsin), and IgG2a was added at a mass ratio of 8: 1 (antibody: Pepsin). After adding the pepsin solution, the mixture was incubated at 37 ° C for 2.5 hours. Then, 1/10 volume of 2 MT ris was added to stop the digestion reaction.

 <Example 25 Antibody Labeling>

For direct labeling of an antibody, generally, an enzyme such as alkaline phosphatase or peroxidase is bound to a reamino group or an SH group by the periodic acid method or the maleimide method. The antibody prepared in Example 12 was subjected to peroxidase labeling kit _SH (Dojin Chemical Co., Ltd.), and the male group method was applied to the SH group according to the usage volume of the manual attached to the kit. Luoxidase labeling was applied.

<Example 26: Comparison of standard curve with conventional measurement system>

 ELISA standard curves were prepared using PTX 3 protein standards (AL EX IS) that had been tested for concentration. 3, 1.1, 0.37, 0.12, 0.041, 0.014, 0. It was prepared using a standard dilution of 005 ng / mL. Furthermore, a standard curve was determined in the same manner as described above using a kit for measuring the patient blood concentration in WO 2005/080981 in order to compare the sensitivity of ELIISA.

 In comparison between the multiple measurement systems prepared this time and the measurement using the antibody used in WO 2005/080981, it was clearly shown in a kit using P PMX 01 04 as the antibody coated on the plate and P PMXO 105 as the labeled antibody. It was confirmed that the sensitivity of the new measurement system was excellent (Fig. 19).

 In addition, while the recognition site of the antibody used in the conventional measurement system was an antibody that recognizes the N-terminal site of the PTX3 molecule, P PMXO 1 04 and P PMX01 05 with the new measurement kit use PT X 3 It is an antibody that recognizes the three-dimensional structure I.

<Example 27 Comparison with Conventional Measurement System (Addition Recovery Test and Reproducibility Test)> As described above, the addition recovery test and the reproducibility test were performed in WO 2005/080981 for comparison of ELISA sensitivity. Using the kit that measured the medium concentration, measurement was performed at the same time, and data for comparative study was collected. Actually, the sample used for the addition recovery test was prepared as follows. In other words, add the antigen to the sample dilution buffer for the reference sample, and add the antigen to the 8 human plasma samples for the control sample to a final concentration of 2, 5, and 10 ng / mL. It was. In the reproducibility test, plasma samples prepared from 6 healthy subjects and plasma samples prepared from 3 patients with unstable angina were measured according to the method described below. The measuring method is as follows. That is, 10 L of the prepared sample was injected into a well injected with 100 L of the sample dilution buffer, and a shaking reaction was performed at room temperature for 1 hour. Next, the plate is washed 5 times with a washing solution (PBS, 0.05% Tween 20), and the labeled antibody solution is added to each well. 100 L was injected into the flask and shaken at room temperature for 1 hour. After the reaction, the plate is washed 5 times with a washing solution, and 100 L of TMB coloring solution (ScyTek Laboratories) is injected into each well, allowed to react at room temperature for 30 minutes, and the reaction stop solution (Scy T ek Laborator ι es) 100 L was injected into each “Nozore” to stop the reaction, and absorbance at a wavelength of 450 ηm was measured with a microplate reader.

As a result, the addition recovery in the new measurement system showed a good recovery rate at both low and high concentrations, whereas the existing measurement system showed a low recovery rate at the low concentration (Table). Ten) . On the other hand, in the reproducibility test, a good CV value was obtained in the new measurement system, whereas in the existing measurement system, the CV value was high when the low concentration of PTX 3 was added, especially when it was not added (Table 11). These results show that the development of a new measurement system has enabled highly sensitive and accurate measurements.

Comparison with conventional measurement system in addition recovery test

 Existing measurement system New measurement system Endogenous addition Measurement value Recovery rate Endogenous addition Measurement value Recovery rate Test PTX3 PTX3 (%) PTX3 PTX3 concentration (%) Material, ng / mL) (ng / mL), ng / mL) (ng / mu i.ng mL) (.ng / mL)

1 0.8 2.00 2.8 85 1.5 2.00 3.6 97

 5.00 5.4 78 5.00 6.5 106

10.00 9.9 87 10.00 11.0 105

2 1.4 2.00 3.5 90 2.5 2.00 4.4 87

 5.00 6.4 85 5.00 7.2 98

10.00 11.5 96 10.00 11.9 103

3 1.1 2.00 3.1 89 1.6 2.00 3.6 93

 5.00 6.5 92 5.00 6.6 106

10.00 12,1 106 10.00 1 1.4 108

4 0.6 2.00 2.4 78 0.7 2.00 2.8 95

 5.00 5.5 85 5.00 5.6 103

10.00 11.3 103 10.00 10.3 106

5 1.9 2.00 3.6 75 2.2 2.00 4.3 92

 5.00 6.2 73 5.00 6.6 92

10.00 10.44 82 10.00 11.0 95

8 1.4 2.00 3.3 83 1.6 2.00 3.6 86

 5.00 6.7 92 5.00 6.1 96

10.00 11.8 100 10.00 11.1 104

7 0.8 2.00 2.8 85 1.5 2,00 3.5 84

 5.00 5.7 83 5.00 6.1 97

10.00 10.8 95 10.00 10.1 94

8 1.1 2.00 3.0 79 1.5 2.00 3.4 83

 5.00 5.9 82 5.00 6.1 98

10.00 10.0 85 10.00 10.7 101 [Table 11] Sample Existing measurement system absorbance value New measurement system absorbance value

 Mean SD CV (¾) Mean SD CV (¾)

Ong / mL 0.014 0.00141 10.1 0.013 0.00055 4.3

0.125ng / mL 0.037 0.00281 7.6 0.035 0.00071 2.0

(U-con 0.25ng / mL 0.063 0.00191 3.0 0.055 0.00084 1.5 lng / L 0.184 0.00630 3.5 0.174 0.00148 0.9 Binant 4ng / iL 0.634 0.05162 8.1 0.623 0.00592 0.9

PTX3) 1 Ong / mL 1.446 0.07283 5.0 1.430 0.01841 1.3

 16ng / mL 2.327 0.02263 1.0 2.172 0.01736 0.8

1 0.147 0.00483 3.3 0.124 0.00195 1.6

2 0.142 0.00636 4.5 0.124 0.00207 1.7 Healthy subject 3 0.130 0.00354 2.7 0.108 0.00217 2.0 Plasma specimen 4 0.282 0.00424 1.5 0.226 0.00336 1.5

 5 0.269 0.01485 5.5 0.224 0.00385 1.7

6 0.145 0.00441 3.0 0.115 0.00365 3.2 Angina 1 0.737 0.02831 3.8 0.594 0.00646 1.1 Plasma specimen 2 0.988 0.02242 2.3 0.825 0.00466 0.6

Claims

The scope of the claims  [1] A method for determining the degree of heart failure and / or prognosis, comprising measuring PTX3 concentration in a test sample using an anti-PTX3 monoclonal antibody. [2] The method according to claim 1, wherein the determination of the prognosis of heart failure is to monitor symptoms of a heart failure patient. [3] The method according to claim 1 or 2, wherein the heart failure is non-ischemic chronic heart failure or idiopathic dilated cardiomyopathy. [4] The determination method according to any one of claims 1 to 3, wherein the test sample is blood, serum or plasma.  [5] The determination method according to any one of [1] to [4], wherein an anti-PTX3 monoclonal antibody immobilized on a support and an anti-PTX3 monoclonal antibody labeled with a labeling substance are used.  [6] The determination method according to any one of claims 1 to 5, wherein the anti-PTX3 monoclonal antibody is a monoclonal antibody or a fragment thereof that recognizes the full-length PTX3 conformational epitope.  [7] Anti-P T X 3 monoclonal antibody is P PMX01 02 (FERM BP—  1 0326), P PMX01 04 (FERM B P- 1 07 1 9) or P PMX 01 05 (FERM BP— 1 0720), The determination method according to any one of claims 1 to 6.  [8] A diagnostic agent for the degree and / or prognosis of heart failure, comprising an anti-PTX 3 monoclonal antibody.  [9] A diagnostic agent for the degree and / or prognosis of heart failure, characterized by measuring PTX 3 concentration in a test sample using an anti-PTX3 monoclonal antibody.  [10] Use of an anti-P T X3 monoclonal antibody for the manufacture of a diagnostic agent for the degree and / or prognosis of heart failure.