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WO2008012941A1 - Méthode de diagnostic d'une insuffisance cardiaque - Google Patents

Méthode de diagnostic d'une insuffisance cardiaque Download PDF

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Publication number
WO2008012941A1
WO2008012941A1 PCT/JP2007/000778 JP2007000778W WO2008012941A1 WO 2008012941 A1 WO2008012941 A1 WO 2008012941A1 JP 2007000778 W JP2007000778 W JP 2007000778W WO 2008012941 A1 WO2008012941 A1 WO 2008012941A1
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WIPO (PCT)
Prior art keywords
ptx
ptx3
antibody
heart failure
protein
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PCT/JP2007/000778
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English (en)
Japanese (ja)
Inventor
Koichi Node
Teruo Inoue
Norihiko Kotooka
Yasuchika Takeishi
Satoshi Suzuki
Isao Kubota
Yukio Ito
Mina Sagara
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Saga University NUC
Perseus Proteomics Inc
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Saga University NUC
Perseus Proteomics Inc
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Publication of WO2008012941A1 publication Critical patent/WO2008012941A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

Definitions

  • the present invention relates to a method for detecting PTX 3 in blood with high sensitivity and determining the degree of heart failure.
  • 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).
  • IL_1 interleukin 1
  • 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.
  • PTX3 is not guided by I L-6.
  • 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).
  • 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.
  • 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.
  • ECG Ischemia is important for the diagnosis of arrhythmia, but there is little information on heart failure itself.
  • Chest X-ray Useful in determining the extent of cardiac enlargement and pulmonary congestion, but difficult to distinguish when present such as pneumonia.
  • Echocardiogram Structure ⁇ Functional aspects can be analyzed.
  • 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.
  • Norepinephrine and renin in blood Effective in severe cases, but it is difficult to distinguish in mild cases.
  • 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.
  • 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.
  • 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.
  • 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)
  • Non-Patent Document 4 Circua latio, 10 02, 636— 4 1 (2000)
  • Patent Document 1 WO 2005-08098 1
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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 ⁇ ⁇ ⁇ ⁇ ⁇ 3 protein prepared in Example 11 and ⁇ 3 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 ⁇ ⁇ X 3 polypeptide in Example 18 and ⁇ ⁇ X 3 sensitized mouse serum as the primary antibody in Example 18 are shown.
  • ⁇ 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 ⁇ ⁇ X 3 in Example 20 and the reactivity with ⁇ ⁇ 01 04.
  • FIG. 18 shows the relationship between the ligation peptide digestion time under non-reducing conditions of full length ⁇ ⁇ X 3 in Example 20 and the reactivity with ⁇ ⁇ 01 05.
  • FIG. 19 A comparison of standard curves of the conventional ELISA kit and the present ELISA kit is shown.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the PTX 3 protein is preferably measured by an immunological assay using an anti-PTX3 antibody.
  • the measurement method using the anti-PTX 3 antibody will be described in detail.
  • 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).
  • 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.
  • 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.
  • 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.
  • the anti-PTX 3 antibody used in the present invention is a polyclonal antibody using known means. Or as a monoclonal antibody.
  • 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.
  • 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.
  • a monoclonal antibody can be prepared as follows.
  • 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.
  • this purified PTX3 protein is used as a sensitizing antigen.
  • a partial peptide of PTX3 can be used as a sensitizing antigen.
  • 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.
  • 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.
  • Immunization of an animal with a sensitizing antigen is performed according to a known method.
  • a common method is to inject a sensitizing antigen intraperitoneally or subcutaneously in a mammal. Is done.
  • 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.
  • 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.
  • immune cells are collected from the mammal and subjected to cell fusion.
  • Preferred immune cells In particular, splenocytes are mentioned.
  • 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.
  • 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.
  • the cell fusion is performed in a normal nutrient culture medium in the presence of, for example, a cell fusion promoter.
  • 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.
  • P EG polyethylene glycol
  • HV J Sendai virus
  • auxiliary agent such as dimethyl sulfoxide
  • the use ratio of immune cells and myeloma cells can be arbitrarily set.
  • the number of immune cells is preferably 1 to 10 times that of myeloma cells.
  • 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.
  • serum supplements such as fetal calf serum (FCS) can be used in combination.
  • 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).
  • PEG polyethylene glycol
  • 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.
  • 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.
  • 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.
  • a HAT culture solution a culture solution containing hypoxanthine, aminopterin and thymidine
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • the former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.
  • 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.
  • antibody fragments include, for example, Fab, Fab ′, F (ab ′) 2, Fv, Diabody, and the like.
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • ELI SA enzyme-free “5” free enzyme-enhanced Mi method
  • the above-described immunological methods such as ELISA can be performed by methods known to those skilled in the art.
  • 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.
  • 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.
  • the binding between the anti-PTX3 antibody and the PTX3 protein is usually performed in a buffer solution.
  • a 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.
  • a buffer containing a surfactant such as Tween20 is used.
  • 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.
  • 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.
  • 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.
  • Labeling of the anti- ⁇ X3 antibody can be performed by a generally known method.
  • 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.
  • 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.
  • 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 '.
  • 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.
  • 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.
  • a substrate is added, and enzymatic changes of the substrate, for example, color development, can be detected with an absorptiometer.
  • 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).
  • ABTS ABTS
  • TMB 5'-tetramethylbenzidine
  • a fluorescent substance it can be detected by a fluorometer.
  • 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
  • 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.
  • 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.
  • the secondary antibody is preferably labeled with a labeling substance.
  • a detection method using an agglutination reaction can be mentioned. In this method, ⁇ X3 can be detected using a carrier sensitized with an anti- ⁇ 3 antibody.
  • any carrier may be used as long as it is insoluble, does not cause a non-specific reaction, and is stable.
  • latex particles bentonite, collodion, kaolin, fixed sheep erythrocytes and the like can be used, but it is preferable to use latex particles.
  • 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.
  • ⁇ X3 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.
  • Another embodiment of the method for measuring ⁇ X3 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.
  • 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.
  • 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.
  • 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.
  • Diagnostic agents include kits.
  • the diagnostic agent may contain a carrier for immobilizing the antibody, and the antibody may be bound to the carrier in advance.
  • a carrier to which an antibody is adsorbed may be included.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 3 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.
  • n 1 34, hereafter referred to as exclusion group.
  • cardiac event means death or readmission due to worsening heart failure.
  • S Table 2 shows the results of a clinical background comparison using tat View Ver. 5.0 (SAS Institute Institute USA).
  • NYHA Cardiac Classification (I 1 II / III / IV) 26/37/23/61/12/24/5 ⁇ 0.0001 Hypertension 44 (483 ⁇ 4) 16 (333 ⁇ 4) 0.2933 Diabetes 21 (233 ⁇ 4) 10 (24%) 0.9003 High Lipemia 20 (223 ⁇ 4) 3 (73 ⁇ 4) 0.1681 Etiology of chronic heart failure 0.4424 Dilated cardiomyopathy 24 (263 ⁇ 4) 15 (363 ⁇ 4)
  • 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
  • PTX3 (ng / niL) 5.3 Sat 5.5 15.2 Sat 26.8 0.0027
  • 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 ⁇ )
  • 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.
  • p 0.
  • 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.
  • CH F chronic heart failure
  • ACEI / ARB Angiotensin post-change II element or angiotensin receptor blocker
  • event group the group in which an event occurred
  • no event group the group in which no event occurred
  • event occurrence means death or readmission due to worsening heart failure.
  • 1 patient died due to CH F exacerbation and 5 patients were readmitted due to CH F exacerbation.
  • Cox multivariate regression analysis SAS institute Ic.
  • Single-strand cDNA of human umbilical vein endothelial cells 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).
  • AMV Reverse Transcr Iptase First—strandc DNA Synthesis Kit manufactured by Seikagaku Corporation.
  • 5'-Amp I i FIN DER RACE Kit CI ontech
  • PC's 5'-RACE method Frohman, MA eta I. , Proc. N at I. A ca d. S ci.
  • 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 ⁇ 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.
  • a buffer solution (20 mM HEP ES, 15 mM NaCI, 0.05% sodium azide, pH 7.2
  • 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.
  • 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.
  • FCA Full's complete adjuvant
  • FIA Freund's incomplete adjuvant, D ifco (0639-60), Becton Dickinson (cat # 26391 0)
  • 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.
  • 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.
  • 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.
  • 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
  • the polypeptide at the N-terminal part of 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.
  • 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.
  • PTX 3 protein obtained in Example 11 and the polypeptide of the N-terminal part of PTX 3 described in Example 15 were used as materials.
  • N—PTX3 polypeptide of the N-terminal part of PTX 3 described in Example 15
  • a general antigen solid-phase ELISA method was used as a method for specifying the binding site. 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.
  • 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.
  • 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.
  • 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).
  • Hybond_ECL GE Healthcare
  • C_PTX3 C-terminal part of PTX3 (positions 1 79 to 381 of SEQ ID NO: 2) in a CHO cell line was performed by the following method.
  • N— N-terminal polypeptide 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.
  • 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.
  • 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).
  • 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.
  • 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 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 ⁇ g 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.
  • 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.
  • 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.
  • 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.
  • P PMX O 104 and P PMX O 105 were antibodies that recognize the three-dimensional structure of PTX3.
  • 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 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).
  • DFP Fluorophosphate Disopropyl
  • CBB Kumashi Brilliant Blue
  • 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.
  • FERM BP— 1 07 1 9 and FE RM BP are FERM BP— 1 07 1 9 and FE RM BP, respectively.
  • the coupling constant was measured using a BIAcor 3000 system (BIAcorre, Uppsala, Sweden).
  • a BIAcor 3000 system (BIAcorre, Uppsala, Sweden).
  • an anti-mouse Ig G antibody was immobilized on the sensor chip CM5 using the NHSS / EDC coupling method.
  • 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 ⁇ g / mL in surfactant P 20) was injected to immobilize an antibody of several hundred RU.
  • 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).
  • 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).
  • a dilute buffer 5 mM Tris—HCI, 15 OmM NaCI, pH 7.5.
  • 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.
  • 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.
  • 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.
  • a washing solution PBS, 0.05% Tween 20
  • 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 ⁇ es) 100 L was injected into each “Nozore” to stop the reaction, and absorbance at a wavelength of 450 ⁇ m was measured with a microplate reader.
  • TMB coloring solution ScyTek Laboratories

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Abstract

L'invention porte sur une méthode de diagnostic d'une insuffisance cardiaque à un stade précoce, et spécifiquement sur une méthode de détermination de l'importance de l'insuffisance cardiaque et/ou de son pronostic, caractérisée par la mesure du niveau de PTX3 dans un échantillon en utilisant un anticorps monoclonal anti-PTX3.
PCT/JP2007/000778 2006-07-25 2007-07-20 Méthode de diagnostic d'une insuffisance cardiaque Ceased WO2008012941A1 (fr)

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US12152070B2 (en) 2018-09-14 2024-11-26 Ohealth Biopharmaceutical (Suzhou) Co., Ltd. Medicinal composition containing monoclonal antibody or antibody fab fragment thereof, and use thereof

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ASO S.: "Koketsuatsu ni yoru Hyoteki Zoki Shogai Hyokaho Handbook -Rinsho Genba deno Katsuyoho- Kokando C Han'nosei Tanpaku o Mochiita Koketsuatsu ni yoru Zoki Shogai no Hyokaho", CARDIOANGIOLOGY, vol. 56, no. 3, 2004, pages 302 - 309, XP003020691 *
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Publication number Priority date Publication date Assignee Title
WO2019056991A1 (fr) * 2017-09-19 2019-03-28 臻崴生物科技有限公司 Anticorps monoclonal ou fragment de liaison à l'antigène de celui-ci, et son utilisation
US12152070B2 (en) 2018-09-14 2024-11-26 Ohealth Biopharmaceutical (Suzhou) Co., Ltd. Medicinal composition containing monoclonal antibody or antibody fab fragment thereof, and use thereof

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