WO2005080981A1 - 血管障害の程度の判定方法 - Google Patents
血管障害の程度の判定方法 Download PDFInfo
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- WO2005080981A1 WO2005080981A1 PCT/JP2005/003029 JP2005003029W WO2005080981A1 WO 2005080981 A1 WO2005080981 A1 WO 2005080981A1 JP 2005003029 W JP2005003029 W JP 2005003029W WO 2005080981 A1 WO2005080981 A1 WO 2005080981A1
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- ptx3
- antibody
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- vascular
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
-
- 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
- G01N2800/328—Vasculitis, i.e. inflammation of blood vessels
Definitions
- the present invention relates to a method for determining the degree of a vascular disorder and a method for screening a therapeutic agent for a vascular disorder.
- PTX3 also known as Pentraxin, Pentaxin, TSG-14, MPTX3, belongs to the pentraxin family discovered as being expressed on human umbilical cord endothelial cells stimulated with interleukin 1 (IL 1) It is a secreted protein (Non-Patent Document 1).
- the pentraxin family includes C reactive proteins (CRP) and serum amyloid P component (SAP) known as inflammatory proteins; ⁇ .
- CRP C reactive proteins
- SAP serum amyloid P component
- Pentraxin is also known as Long Pentraxin, and CRP is also called Short Pentraxin.
- Pentraxin is a name derived from having a CRP sequence in its structure, and is presumed to function as an inflammatory protein.
- PTX3 is not induced by IL-6.
- cell types that express the PTX3 protein are also different from CRP and SAP, suggesting that PTX3 also has a different function from CRP and SAP (Non-Patent Document 2).
- inflammation covers a wide range and includes dermatitis, inflammation of various organs, and the like. Among them, vascular inflammation leads to serious diseases such as heart disease and brain disease.
- risk factors for acute myocardial infarction include high blood total cholesterol, hypertension, diabetes, obesity, and smoking.
- high total cholesterol in blood not only diet therapy but also various statins are used as hypolipidemic agents to eliminate this risk factor.
- Brain diseases include dementia due to vascular disorders, and treatment with aspirin Has been done.
- Non-Patent Document 3 As a conventional method for measuring PTX3, a method based on the ELISA method described in Non-Patent Document 3 is known. In this assay, the PTX3 concentration reached a maximum of 22 ng / mL at 7.5 hours after a myocardial infarction attack, and then decreased sharply to a level of 0.5 in normal subjects who did not appear to have heart disease. 2. It is stated that it will be 5 ngZmL. As described above, according to the conventional PTX3 measurement results, it is known that the PTX3 concentration in the blood is increased only during a myocardial infarction attack. I don't really know how the concentration changes!
- Statin and antipyretic analgesic aspirin which are therapeutic agents for vascular disorders, are known as therapeutic agents for vascular disorders. Epidemiologic studies have shown that they improve vascular disorders. There are no drugs developed for the treatment of vascular disorders.
- Non-patent literature l Breviorio et al .: J. Biol. Chem., 267 (31), 22190-7 (1992)
- Non-Patent Document 2 J. Biol. Chem., 267 (31), 22190-7 (1992); Domyaku Koka (Arteriosclerosis), 24 (7-8), 375-80 (1996)
- Non-Patent Document 3 Arthritis and Rheumatism, 44/12 (2841-50), 2001, Circulation, 102, 636-41 (2000), Arterioscler Thromb Vase Biol. 2002; 22: el0-el4
- the degree of vascular damage before myocardial infarction or cerebrovascular dementia can be determined, the degree of heart disease or cerebrovascular disease can be diagnosed, and the therapeutic effect of a therapeutic agent for heart disease or a therapeutic agent for cerebrovascular disease can be determined. Can be determined. In addition, if a system that can determine the degree of vascular disorders can be developed, screening for therapeutic drugs for vascular disorders will be possible.
- an object of the present invention is to provide a method for determining the degree of vascular injury as a risk factor for myocardial infarction and cerebrovascular dementia and a method for screening a therapeutic agent for vascular injury.
- PTX3 concentration which was considered to increase only in the acute phase during a stroke of myocardial infarction, and studied statins and aspirin known to treat vascular disorders.
- the blood PTX3 protein concentration was measured before and after their administration, and the PTX3 concentration was clearly reduced after administration of these drugs.
- the measurement of plasma PTX3 concentration in patients with myocardial infarction and stable angina and unstable angina, which is considered to be a precursor disease showed that PTX3 concentration was clearly higher in proportion to the severity of vascular disorders.
- PTX3 is a diagnostic marker having high specificity for blood vessels, which is not a diagnostic marker for a systemic inflammatory response, and is useful for knowing the degree of injury.
- this method can be used as a screening method for a therapeutic drug for vascular disorders, and completed the present invention.
- a new anti-PTX3 antibody was prepared and a high-sensitivity measurement system was established, and PTX3 in serum was measured before and after statin administration and before and after aspirin administration, and a decrease in PTX3 after administration was confirmed.
- the present invention has been completed.
- the present invention provides a method for determining the degree of vascular injury, which comprises measuring the concentration of PTX3 in a test sample.
- the present invention also provides a diagnostic agent for the degree of vascular disorder containing an anti-PTX3 antibody.
- the present invention provides a method for screening a therapeutic agent for vascular disorders, which comprises administering or contacting a test substance to an animal or a cell, and measuring a change in PTX3 protein or PTX3 gene amount.
- the present invention it is possible to diagnose the degree of vascular damage before the onset of acute myocardial infarction, vascular dementia, etc., thereby diagnosing the degree of heart disease or cerebrovascular disease, and And a shift to brain disease can be prevented. Also, blood vessels due to drug treatment Provides guidance on treatment of disorders. In addition, new therapeutic agents for cardiovascular and cerebrovascular disorders can be screened.
- FIG. 1 is a diagram showing the results of comparison between recombinant PTX3 and native PTX3 by Western blotting.
- FIG. 2 is a view showing a plurality of newly constructed ELISA standard curves.
- FIG. 3 is a diagram showing a comparison between a newly constructed PTX3 measurement system provided for clinical sample measurement and a conventional PTX3 measurement system.
- FIG. 4 is a view showing the measurement results of PTX3 in the blood of a heart disease patient.
- the measurement includes a quantitative or non-quantitative measurement.
- the non-quantitative measurement it is simply determined whether or not the PTX3 protein is present, or the PTX3 protein is present in a certain amount or more. Measurement, comparing the amount of PTX3 protein with other samples (for example, control samples), and the like.
- Quantitative measurement includes measurement of PTX3 protein concentration, PTX3 protein And the like can be used.
- the nucleotide sequence of the PTX3 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the PTX3 protein is shown in SEQ ID NO: 2.
- the test sample is not particularly limited as long as it is a sample that may contain the protein of PTX3, but a sample obtained by collecting the physical strength of an organism such as a mammal is preferable, and a sample obtained from human is more preferable.
- Sample Specific examples of the test sample include, for example, blood, interstitial fluid, plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph, saliva, urine, etc. Preferred are blood, serum and plasma.
- a sample obtained from a test sample such as a culture solution of cells collected from an organism, is also included in the test sample of the present invention.
- Vascular damage is a process that involves damage to vascular endothelial cells, migration of vascular medial smooth muscle cells, accumulation of macrophages, foaming, thrombus adhesion, plaque formation, vascular fibrosis, and plaque rupture. And severe disease.
- vascular disorders in the present invention include hyperlipidemia and brain disease, as well as hypertension. Includes vascular disorders caused by disease, diabetes, obesity and smoking.
- the degree of the vascular disorder refers to the degree of the disorder accompanying the progression of the vascular disorder. That is, the vascular disorder following the above-mentioned progression process is histopathologically shown as a measure of the extent to which the plaque ruptures finally. (a) the size of the lipid core, (b) the thickness of the fibrous cap, (c) the intensity of shear stress, (d) the degree of infiltration of inflammatory cells, and (a) the larger the The thinner (b), the stronger (c), and the stronger (d), the more plaque-bursting is. Therefore, the degree of vascular damage in the present invention refers to the above (a) to (d).
- the measurement of PTX3 is preferably an immunoassay using an anti-PTX3 antibody.
- the measurement method using the anti-PTX3 antibody will be described in detail.
- the anti-PTX3 antibody used in the present invention may be of any type as long as it specifically binds to the PTX3 protein, regardless of its origin, type (monoclonal or polyclonal) and shape.
- known antibodies such as a mouse antibody, a rat antibody, a human antibody, a chimeric antibody, and a humanized antibody 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-PTX3 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means.
- a mammal-derived monoclonal antibody is particularly preferable.
- Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques.
- a monoclonal antibody-producing hybridoma can be basically produced as follows using a known technique. That is, PTX3 is used as a sensitizing antigen, immunized with the usual immunization method, the obtained immunocytes are fused with a known parent cell by a normal cell fusion method, and monoclonal antibodies are obtained by a normal screening method.
- Antibody-producing cells It can be prepared by screening.
- a monoclonal antibody may be prepared as follows.
- PTX3 which is used as a sensitizing antigen for obtaining an antibody, is obtained by purifying the available cell culture supernatant. Alternatively, it can be obtained according to the method disclosed in JP-T-2002-503642.
- 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 PTX3, or can be obtained by incorporating a part of the PTX3 gene into an expression vector, and further, natural PTX3 can be decomposed by a protease. Can also be obtained.
- the portion and size of PTX3 used as a partial peptide are not limited.
- the mammal to be immunized with the sensitizing antigen is not particularly limited, but it is generally preferable to select a mammal in consideration of compatibility with the parent cell used for cell fusion. Rodents animals, for example, mice, rats, genotypes, musters, or egrets and monkeys are used.
- Immunization of an animal with a sensitizing antigen is performed according to a known method.
- the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal.
- the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and then mixed with an appropriate amount of a normal adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified.
- a suitable carrier can be used at the time of immunization with the sensitizing antigen.
- a partial peptide having a small molecular weight is used as a sensitizing antigen, it is preferable to immunize albumin or keyhole limpet by binding to a carrier protein such as mosine.
- the mammal After immunizing a mammal in this way and confirming that the level of the desired antibody in the serum increases, the mammal is also harvested for immune cells and subjected to cell fusion.
- Preferred immune cells are And especially splenocytes.
- Mammalian myeloma cells are used as the other parent cells to be fused with the immune cells.
- the myeloma cells can be obtained from various known cell lines, for example, P3 (P3x63Ag8.653) ( J. Immnol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M.
- the cell fusion of the immune cells and myeloma cells is basically performed by a known method, for example, the method of Kohler and Milstein, et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73 , 3-46).
- the cell fusion is carried out, for example, in a normal nutrient medium in the presence of a cell fusion promoter.
- a cell fusion promoter for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if necessary, an auxiliary agent such as dimethyl sulfoxide can be added and used to enhance the fusion efficiency.
- the ratio of the use of the immune cells to the myeloma cells can be arbitrarily set. For example, it is preferable that the number of immune cells be 110 to 10 times that of myeloma cells.
- the culture medium used for the cell fusion for example, an RPMI1640 culture medium, a MEM culture medium suitable for the growth of the myeloma cell line, and other ordinary culture mediums used for this kind of cell culture can be used. Further, a serum replacement solution such as fetal calf serum (FCS) can be used in combination.
- FCS fetal calf serum
- polyethylene glycol (for example, having an average molecular weight of 1,000 to 6000) is prepared by mixing a predetermined amount of the immune cells and myeloma cells in the culture solution and heating the mixture to about 37 ° C in advance.
- the solution is usually added at a concentration of 30-60% (w / v) and mixed to form the desired fused cells (hybridomas).
- an operation of successively adding an appropriate culture medium and centrifuging to remove the supernatant is repeated to remove the cell fusion agent and the like which are favorable for the growth of the hybridoma.
- the thus obtained hybridoma can be used in a usual selective culture medium, for example, a HAT culture medium.
- a culture solution containing hypoxanthine, aminopterin and thymidine Selected.
- the culturing in the HAT culture solution is continued for a time (usually several days to several weeks) sufficient for the death of cells (non-fused cells) other than the target hybridoma.
- the usual limiting dilution method is performed, and screening and single-cloning of the hybridoma producing the desired antibody are performed.
- Screening and single-cloning of the desired antibody may be performed by a screening method based on a known antigen-antibody reaction.
- 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, washed with the carrier, and reacted with an enzyme-labeled secondary antibody, etc.
- a hybridoma producing the desired antibody can be cloned by a limiting dilution method or the like. At this time, the antigen used for immunization may be used.
- the thus-produced monoclonal antibody producing hybridomas can be subcultured in a normal culture solution, and can be stored in liquid nitrogen for a long period of time. .
- the hybridoma is obtained by culturing the hybridoma according to a usual method and obtaining a culture supernatant thereof, or administering the hybridoma to a mammal compatible therewith. Then, a method of obtaining the ascites is used.
- the former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.
- the antibody gene is cloned into a hybridoma.
- a recombinant vector can be used, which is inserted into an appropriate vector, introduced into a host, and produced using a gene recombination technique (for example, Vandamme, AM et al., Eur. J Biochem. (1990) 192, 767-775, 1990).
- mRNA encoding the variable (V) region of the anti-PTX3 antibody is isolated from the hybridoma producing the anti-PTX3 antibody.
- the mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159) is used to prepare total RNA, and then the desired mRNA is prepared using mRNA Purification Kit (Pharmacia). Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).
- cDNA of the antibody V region is synthesized from the obtained mRNA using reverse transcriptase. Synthesis of cDNA is performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation) or the like. In order to synthesize and amplify cDNA, the 5, -RACE method using the 5,-Ampli FINDER RACE Kit (Clontech) and PCR (Frohman, MA et al., Proc. Natl. Acad. Scad. USA (1988) 85, 8 998-9002, Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) and the like can be used.
- a target DNA fragment is purified from the obtained PCR product and ligated to a vector DNA.
- a recombinant vector is prepared from this, introduced into E. coli or the like, and colonies are selected to prepare a desired recombinant vector. Then, the base sequence of the target DNA is confirmed by a known method, for example, a dideoxynucleotide chain termination method or the like.
- the DNA is inserted into an expression vector containing the DNA encoding the desired antibody constant region (C region).
- the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region, for example, an enhancer or a promoter.
- an expression control region for example, an enhancer or a promoter.
- host cells are transformed with this expression vector to express the antibody.
- Antibody gene expression is determined by separating the DNA encoding the antibody heavy chain (H chain) or light chain (L chain).
- the host cells may be co-transformed by incorporating them into an expression vector, or the host cells may be transformed by incorporating DNA encoding the H chain and L chain into a single expression vector (W094Z11523). No.).
- transgenic animals that can use not only the above host cells but also can be used.
- an antibody gene is inserted into a gene encoding a protein (eg, goat ⁇ -casein) that is specifically produced in milk to prepare a fusion gene.
- a DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the embryo is introduced into a female goat.
- Embryo-accepted goats The milk produced by the born transgeneic goat or its offspring also produces the desired antibodies.
- Hormones may also be used in transgenic enigma as appropriate in order to increase the amount of milk containing the desired antibody that is also produced by transgenic energetics (Ebert, KM et al., Bio / Technology (1 994) 12, 699—702).
- artificially modified recombinant antibodies for example, chimeric antibodies and humanized antibodies can be used. These modified antibodies can be produced using known methods.
- the chimeric antibody is produced by ligating the DNA encoding the antibody V region obtained as described above with the DNA encoding the human antibody C region, incorporating the DNA into an expression vector, introducing the resulting vector into a host, and producing the chimeric antibody. Obtained from this. Using this known method, a chimeric antibody useful in the present invention can be obtained.
- the humanized antibody is also referred to as a reshaped human antibody, which is used to replace the complementarity determining region (CDR) of a non-human mammal, for example, a mouse antibody. It has been transplanted into the complementarity determining region, and its general gene recombination technique is also known (see European Patent Application Publication No. EP 125023, W096Z02576).
- a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody has a portion overlapping both terminal regions of both the CDR and FR.
- FR framework region
- a region in which the complementarity determining region forms a favorable antigen-binding site is selected. If necessary, the amino acids of the framework region in the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (19 93) 53, 851— 856) Q
- C region of the chimeric antibody and the humanized antibody those of a human antibody are used.
- C ⁇ 1, C ⁇ 2, C ⁇ 3, and C ⁇ 4 are used for the H chain, and C ⁇ , C ⁇ can be used.
- the human antibody C region may be modified in order to improve the stability of the antibody or its production.
- a chimeric antibody works with the variable region of an antibody derived from a mammal other than human and the constant region derived from a human antibody.
- a humanized antibody is composed of a complementarity determining region of an antibody derived from a mammal other than human, a framework region and a C region derived from a human antibody. Since the humanized antibody has reduced antigenicity in the human body, it is useful as an active ingredient of the therapeutic agent of the present invention.
- the antibody used in the present invention is not limited to the whole antibody molecule, and includes a bivalent antibody and a monovalent antibody, which may be a fragment of the antibody or a modified product thereof, as long as it binds to antibody 3.
- Fab, F (ab ') 2 Fv, Fab Zc having one Fab and complete Fc, or Fv of H chain or L chain were linked with an appropriate linker.
- the antibody is treated with an enzyme such as papain or pepsin to generate an antibody fragment, or a gene encoding these antibody fragment is constructed and introduced into an expression vector.
- an enzyme such as papain or pepsin
- the scFv is obtained by linking the H chain V region and the L chain V region of the antibody.
- the H chain V region and L chain V region are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 8 5, 5879—5883).
- a linker preferably a peptide linker
- the H chain V region and L chain V region in the scFv are described herein as antibodies, they may be derived from the deviation.
- the peptide linker connecting the V regions for example, any single-chain peptide having 12 to 19 amino acid residues can be used.
- the DNA encoding the scFv may be all or all of the sequences of the DNA encoding the H chain or H chain V region and the DNA encoding the L chain or L chain V region of the antibody.
- the DNA portion encoding the desired amino acid sequence is made into a type II, amplified by the PCR method using a pair of primers defining both ends thereof, and then the DNA encoding the peptide linker portion, and both ends thereof are each H chain, It is obtained by combining and amplifying a primer pair that is defined so as to be linked to the L chain.
- DNAs encoding scFv are prepared, an expression vector containing them and a host transformed by the expression vector can be obtained according to a conventional method. By using, scFv can be obtained according to a conventional method.
- Antibodies in the present invention also include fragments of these antibodies.
- an anti-PTX3 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.
- the antibody used in the present invention may be a bispecific antibody.
- the bispecific antibody may be a bispecific antibody having an antigen-binding site that recognizes a different epitope on the PTX3 molecule, or one antigen-binding site may recognize PTX3 and the other antigen-binding site may A labeling substance or the like may be recognized.
- Bispecific antibodies It can be prepared by binding HL pairs of two types of antibodies, or can be obtained by fusing hybridomas producing different monoclonal antibodies to produce a bispecific antibody-producing fusion cell. Furthermore, bispecific antibodies can be produced by genetic engineering techniques.
- the antibody gene constructed as described above can be expressed and obtained by a known method.
- expression can be achieved by operably linking a useful promoter commonly used, an antibody gene to be expressed, and a polyA signal downstream of the 3 ′ side thereof.
- a useful promoter commonly used for example, there can be mentioned human cytomegalovirus wax Ino-less early promoter / En'no ⁇ capacitors 1 ⁇ ⁇ the (human cytomegalovirus immediate early promoter / en hancer).
- promoters that can be used for antibody expression used in the present invention, such as retroviruses, polioviruses, adenoviruses, Simian virus 40 (SV40), and other promoters such as Winenores promoters Z enhancers, Some include promoters derived from mammalian cells, such as human cell aging factor-1 la (HEFla).
- the gene can be easily expressed by the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322).
- the gene can be expressed by operably linking a useful promoter commonly used, a signal sequence for antibody secretion, and an antibody gene to be expressed.
- the promoter include a lacz promoter and an araB promoter.
- the lacz promoter use the method of Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422—2427) [From here, use the araB promoter! In this case, it can be expressed by the method of Better et al. (Science (1988) 240, 1041-1043).
- a pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4379) is used for production in the periplasm of Escherichia coli. do it. After separating the antibody produced in the periplasm, the antibody structure is appropriately refolded and used.
- the vector can include aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, large S bacterium xanthinguanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, etc. as selectable markers. .
- APH aminoglycoside transferase
- TK thymidine kinase
- Ecogpt large S bacterium xanthinguanine phosphoribosyltransferase
- dhfr dihydrofolate reductase
- Eukaryotic cells include, for example, established mammalian cell lines, insect cell lines, animal cells such as filamentous fungal cells and yeast cells, and prokaryotic cells include, for example, bacterial cells such as E. coli cells.
- Eukaryotic cells include, for example, established mammalian cell lines, insect cell lines, animal cells such as filamentous fungal cells and yeast cells, and prokaryotic cells include, for example, bacterial cells such as E. coli cells.
- the antibody used in the present invention is expressed in mammalian cells, for example, CHO, COS, Kazuma Mie, BHK, Vero, HeLa cells.
- the transformed host cells are cultured in vitro or in vivo to produce the desired antibody.
- Culture of the host cell is performed according to a known method.
- DMEM, MEM, RPMI1640, IMDM can be used as a culture solution, and a serum supplement such as fetal calf serum (FCS) can be used in combination.
- FCS fetal calf serum
- the antibody expressed and produced as described above can be separated from cells and host animals and purified to homogeneity. Separation and purification of the antibody used in the present invention can be performed using an affinity column.
- affinity column For example, columns using a protein A column include Hyper D, POROS, Sepharose F.F. (Pharmacia), and the like.
- antibodies can be separated and purified by appropriately selecting and combining chromatography columns other than the above-mentioned affinity columns, filters, ultrafiltration, salting out, dialysis, etc. (Antibodies A Laboratory Manual. Ed. Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).
- the PTX3 to be measured in the present invention is not particularly limited.
- the method for detecting the PTX3 protein contained in the test sample is not particularly limited, but is preferably detected by an immunological method using an anti-PTX3 antibody.
- the immunological method include, for example, radioimmunoassay, enzymatic immunoassay, fluorescent immunoassay, luminescence immunoassay, immunoprecipitation, immunoturbidimetry, western plot, immunostaining, immunodiffusion, and the like.
- Enzymimnoassay is preferred, and particularly preferred is enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay: £, LISA) (eg, sandwich ELISA).
- Enzyme-linked immunosorbent assay £, LISA
- sandwich ELISA sandwich ELISA
- an anti-PTX3 antibody is immobilized on a support, a test sample is added thereto, followed by incubation, and binding of the anti-PTX3 antibody and the PTX3 protein. After that, washing is performed, and a PTX3 protein bound to the support via an anti-PTX3 antibody is detected to detect a PTX3 protein in the test sample.
- the support used in the present invention includes, for example, insoluble polysaccharides such as agarose and cellulose, synthetic resins such as silicone resin, polystyrene resin, polyacrylamide resin, nylon resin, and polycarbonate resin; An insoluble support such as glass can be used.
- These supports can be used in the form of beads or plates. In the case of beads, a column or the like filled with these can be used. In the case of a plate, a multiwell plate (eg, a 96-well multiwell plate) or a biosensor chip can be used.
- the binding between the anti-PTX3 antibody and the support can be achieved by a commonly used method such as chemical bonding or physical adsorption. All of these supports can be used commercially.
- the binding between the anti-PTX3 antibody and the PTX3 protein is usually performed in a buffer.
- a buffer for example, a phosphate buffer, a Tris buffer, a citrate buffer, a borate buffer, a carbonate buffer, and the like are used.
- Incubation conditions include those already used, such as incubation at 4 ° C and room temperature for 1 hour to 24 hours. Is done. Washing after the incubation may be performed using anything that does not prevent 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 provided in addition to the test sample from which PTX3 protein is to be detected.
- Control samples include a negative control sample containing no PTX3 protein and a positive control sample containing PTX3 protein.
- it is possible to detect the PTX3 protein in the test sample by comparing the result obtained with the negative control sample containing no PTX3 protein with the result obtained with the positive control sample containing the PTX3 protein. is there.
- a series of control samples in which the concentration is changed stepwise are prepared, the detection results for each control sample are obtained as numerical values, a standard curve is created, and the test sample It is also possible to quantitatively detect the PTX3 protein contained in the sample.
- a preferable example of the measurement of PTX3 protein bound to a support via an anti-PTX3 antibody is a method using an anti-PTX3 antibody labeled with a labeling substance.
- a test sample is brought into contact with an anti-PTX3 antibody immobilized on a support, and after washing, detection is performed using a labeled antibody that specifically recognizes the PTX3 protein.
- the labeling of the anti-PTX3 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, a radioactive substance, and specific examples include radioisotopes ( 32 P, 14 C, 125 I, 3 H, 131 I, etc.), fluorescein, rhodamine, dansylcotide lid, umberifferon, luciferase, peroxidase, alkaline phosphatase, ⁇ -galactosidase, j8-darcosidase, horseradish peroxidase, glucomirror And lysozyme, saccharidoxidase, microperoxidase, biotin and the like.
- biotin When using biotin as a labeling substance, it is preferable to add a biotin-labeled antibody and then add avidin to which an enzyme such as alkaline phosphatase is bound.
- an enzyme such as alkaline phosphatase
- a known method such as the dartalaldehyde method, the maleimide method, the pyridyl disulfide method, and the periodate method can be used. it can.
- a solution containing an anti-PTX3 antibody is placed on a support such as a plate, and the anti-PTX3 antibody is immobilized on the support.
- the plate block with, for example, BSA, gelatin, albumin, etc. to prevent non-specific binding of proteins. Wash again and place the test sample on a plate.
- wash and add labeled anti-PTX3 antibody After appropriate incubation, wash the plate and detect any remaining labeled anti-PPTX3 antibody on the plate.
- Detection can be performed by a method known to those skilled in the art. For example, in the case of labeling with a radioactive substance, detection can be performed by liquid scintillation or the RIA method.
- a substrate is added, and enzymatic change of the substrate, for example, color development can be detected by an absorptiometer.
- the substrate include 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammo-dum salt (ABTS), 1,2-phenylene-diamine (orsofu-dienamine), 3, 3 ', 5, 5-tetramethylbenzidine (TME) and the like.
- TME 5-tetramethylbenzidine
- a fluorescent substance it can be detected by a fluorometer.
- a particularly preferred embodiment of the method for measuring PTX3 protein of the present invention includes a method using an anti-PTX3 antibody labeled with biotin and avidin.
- a solution containing an anti-PTX3 antibody is placed on a support such as a plate, and the anti-PTX3 antibody is immobilized. After washing the plate, block with, for example, BSA to prevent non-specific binding of proteins. Wash again and add the test sample to the plate. After the incubation, the plate is washed and a biotin-labeled anti-PTX3 antibody is removed. After an appropriate incubation, the plate is washed, and avidin conjugated with an enzyme such as alkaline phosphatase or peroxidase is removed. After the incubation, wash the plate, add the substrate corresponding to the enzyme bound to avidin, and detect the PTX3 protein based on the enzymatic change of the substrate.
- an enzyme such as alkaline phosphatase or peroxidase
- one or more primary antibodies that specifically recognize the PTX3 protein and one or more secondary antibodies that specifically recognize the primary antibody are used. Methods can be mentioned.
- a test sample is contacted with one or more kinds of anti-PTX3 antibodies immobilized on a support, After incubation, washing is performed, and the bound PTX3 protein is detected by the primary anti-PTX3 antibody and one or more secondary antibodies that specifically recognize the primary antibody.
- the secondary antibody is preferably labeled with a labeling substance.
- Another embodiment of the method for measuring the PTX3 protein of the present invention includes a detection method utilizing an agglutination reaction.
- PTX3 can be detected using a carrier sensitized with an anti-PTX3 antibody.
- a carrier for sensitizing the antibody any carrier may be used as long as it is insoluble, does not cause nonspecific reaction, and is stable.
- latex particles bentonite, collodion, kaolin, fixed sheep erythrocytes and the like can be used, but latex particles are preferably used.
- the latex particles for example, it is preferable to use polystyrene latex particles that can use polystyrene latex particles, styrene-butadiene copolymer latex particles, polyvinyl toluene latex particles, and the like.
- the sensitized particles are mixed with the sample and stirred for a certain time.
- the higher the concentration of the anti-PTX3 antibody in the sample the greater the degree of aggregation of the particles. Therefore, PTX3 can be detected by visually observing the aggregation.
- the turbidity due to aggregation can be detected by measuring with a spectrophotometer or the like.
- Another embodiment of the method for measuring the PTX3 protein of the present invention includes, for example, a method using a Noo sensor utilizing the surface plasmon resonance phenomenon.
- a biosensor using the surface plasmon resonance phenomenon can observe the interaction between proteins in real time as a surface plasmon resonance signal using a small amount of protein and without labeling.
- BIAcore Pharmacia
- a test sample is brought into contact with a sensor chip on which an anti-PTX3 antibody is immobilized, and PTX3 protein binding 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 devices, and it is possible to inspect a large number of samples at once.
- the present invention also aims to provide a diagnostic agent for the degree of vascular disorder, but the diagnostic agent contains at least an anti-PTX3 antibody.
- the diagnostic agent includes a kit.
- the diagnostic agent is an ELISA
- the antibody may contain a carrier for immobilizing the antibody, and the antibody may be bound to the carrier in advance.
- the carrier may contain a carrier to which an antibody is adsorbed.
- the diagnostic agent may appropriately contain a blocking solution, a reaction solution, a reaction stop solution, a reagent for processing a sample, and the like.
- a screening method for a therapeutic agent for vascular disorders is carried out by administering or contacting a test substance to an animal or a cell and measuring the amount of PTX3 protein or the amount of PTX3 gene.
- the test substance is administered orally or intravenously, and the blood PTX3 protein or PTX3 gene concentration before and after the administration is measured and compared with the test substance non-administration group.
- animal models of hyperlipidemia, brain disease, diabetes, obesity, and hypertension can be used.
- high-fat diet-loaded animals WHHL ⁇ egrets, apoE-deficient mice, LDL receptor-deficient mice, human-type apoB-introduced mice, dominant mutant apoE-introduced mice, hyper-LDLemia pigs and mi- Pigs and brain disease models are SHRSP rats, and obesity 'diabetes models are leptin-deficient mice, NOD mice, KK / Ta Jcl mice, KK AyZTa Jcl mice, C57BL / KsJ-db / db Jcl mice, C57BL / KsJ- It is preferable to use db / + m Jcl mice, GK / Jcl rats, model animals having vascular disorders such as SHR rats as a model for developing hypertension, and coronary artery balloon injury models (rats, egrets) as a heart disease. Good.
- test substance When cells are used, the test substance is cultivated and cultured using a PTX3 producing cell line and a PTX3 forced expression cell, and the amount of the produced PTX3 protein or PTX3 gene is measured by a known method. After that, screening can be performed by comparing the amount with no addition.
- a known method such as ELISA can be used as a method for measuring the PTX3 protein.
- Examples of the method for measuring the amount of a gene include a method using quantitative RT-PCR.
- a commonly used reporter gene assay can also be used.
- the general method is to use the promoter region of the PTX3 gene with a marker such as luciferase ⁇ galatatosidase, GFP, etc.
- a vector connected upstream of the protein cDNA is prepared, and this vector is transfected into animal cells by a general method.
- the test compound can be screened by adding the test substance to the transfected cells, culturing the cells, and detecting the marker protein by an appropriate method.
- a single-stranded cDNA of human umbilical vein endothelial cell (HUVEC) was prepared by the method described in (0034) above, and was used as a template to design a primer PTX3-F (Kpnl) (sequence based on GenBank number (NM-002852)). No. 3) and ⁇ 3-R (BamHI) (SEQ ID NO: 4) were used to isolate the full-length ORF gene by PCR.
- the fragment obtained by the PCR method was inserted into a vector using the Zero Blunt TO PO PCR Cloning Kit, the nucleotide sequence was analyzed by a standard method, and the fragment cut at the Kpnl site and the BamHI site was converted to a phCMV vector (Stratagene). And transfer vector phCMV-PTX3 was prepared.
- lxl0 5 cells of CHO cells were seeded on a 6-well dish the day before transfection, and cultured, and the following day, 8 ⁇ g of the expression vector was expressed.
- phCMV-3 and 16 ⁇ L of FuGENE 6 reagent were mixed with 100 ⁇ L of serum-free DMEM medium, incubated at room temperature for 20 minutes, and added to the cells.
- clawing was performed using the ultra-dilution method and G418, a selective reagent.
- the culture supernatant of each clone was recovered, and cells expressing the ⁇ 3 protein were screened.
- a clone hereinafter, CHO-PTX3 that constantly expressed about 2-3 gZmL of the PTX3 protein could be selected.
- the protein was purified by the method of Bottazzi et al. (Bottazzi B, Vouret—Craviari, et al., J Biol Chem. 1997; 272 (52): 32817-23.) Specifically, after culturing CHOMA—PTX3 in a 150 cm 2 flask, use a roller bottle (BD Bioscience) and use 300 mL of serum-free medium (S—SFM——, GIBCOZlnvitrogen) per bottle. The rolling speed was adjusted so that the number of revolutions became 1 rotation per minute, and the cells were cultured for 4 days, and the culture supernatant was collected.
- 1 L of the culture supernatant was concentrated to 50 mL using an ultrafiltration membrane concentrator, pellicon XL device Biomax 100 (MILLIPORE).
- the concentrate was dialyzed against 5 L of 50 mM Imidaole, pH 6.6 buffer. The dialysis operation was performed twice on the same buffer solution. It was then applied to ion exchange chromatography using HiPrep 16/10 Q XL (Pharmacia Biotech, Uppsala, Sweden). After washing until the background level was reduced, the concentration of NaCl was increased from 0 to 0.58M for 35 minutes to increase the calorie, and then PTX3 was eluted with 1M NaCl. The elution was monitored at an absorbance of 280 nm.
- Fractions containing the PTX3 protein were collected, and subjected to Genole filtration chromatography using Sephacry S-300 in PBS. Furthermore, in order to obtain only the multimeric form PTX3 of PTX3, purification was performed using a Superose 6 column (Pharmacia Biotech). That is, after performing calibration using a molecular weight standard, PTX3 was applied to the column, and elution was performed with PBS at a flow rate of 0.4 mL Zmin. Each eluted fraction was analyzed by SDS-PAGE to obtain a purified recombinant multimeric PTX3 protein.
- Comparison of natural # 3 and recombinant # 3 was performed by Western blotting. That is, 10 ⁇ L of a 0.5 ⁇ g ZmL solution of natural ⁇ 3 and recombinant ⁇ 3 was subjected to SDS-PAGE in a reduced state, and the gel after electrophoresis was transferred to a PVDF membrane. P on the transferred membrane TX3 was reacted with an anti-PTX3 polyclonal antibody (ALEXIS). Thereafter, the anti-PTX3 polyclonal antibody was reacted with an HRP-labeled anti-Egret IgG antibody, and a PTX3 band was detected. The results are shown in FIG. As shown in FIG. 1, the two were almost identical in molecular weight. It was assumed that the slight difference was due to the difference in sugar chains.
- ALEXIS anti-PTX3 polyclonal antibody
- the monoclonal antibody was prepared by the following operation. That is, BalbZC mice (CRL) or PTX3 knockout mice were immunized with PTX3.
- PTX3 knockout mice were immunized with PTX3.
- For the first immunization prepare immunoprotein at 10 or 100 ⁇ g / animal and use FCA (Freund's complete adjuvant (H37 Ra), Difco (3113-60), betaton Dickinson (cat # 231131)). The emulsion was administered subcutaneously. Two weeks later, 5 or 50 gZ animals were emulsified with FIA (Incomplete Freund's Adjuvant, Difco (0639-60), Betaton Dickinson (cat # 263910)) and subcutaneously administered. Thereafter, booster immunization was performed twice in total at weekly intervals.
- mice were diluted in PBS and administered into the tail vein.
- the mouse myeloma cells P3U1 and mouse spleen cells were mixed, and PEG1500 (Roche's diagnostic, cat # 783 641).
- the cells were inoculated on a 96-well culture plate, and the culture supernatant was screened by ELISA after selection on the HAT medium from the next day.
- the positive clone was subjected to monocloning by the limiting dilution method, and then subjected to expansion culture to collect a culture supernatant. Screening by ELISA was performed using the binding activity to the PTX3 protein as an index, and many anti-PTX3 antibodies having strong and binding ability were obtained.
- the monoclonal antibody was purified using Hi Trap Protein G HP (Amersham CAT # 17-0404-01). Was charged directly to column High Priestess dormer culture supernatant, and eluted with binding buffer (20mM sodium phosphate (P H7. 0)) After washing with elution buffer (0. 1M glycine HCl (pH2. 7)). The eluate was collected in a tube containing a neutralization buffer (1 M Tris-HCl (pH 9.0)) and immediately neutralized. After the antibody eluted fraction was bullated, it was dialyzed all day and night with 0.05% Tween20ZPBS to replace the buffer. The purified antibody was stored at 4 ° C. after adding NaN to 0.02%. [0091] Isotyping of the anti-PTX3 monoclonal antibody was performed as follows.
- Antibody concentrations were determined by performing a mouse IgG sandwich ELISA using goat anti-mouse IgG (gamma) (ZYMED CAT # 62-6600) and alga lipophosphatase-one goat anti-mouse IgG (gamma) (ZYMED CAT # 62-6622).
- a commercially available purified mouse IgGl antibody ZYMED CAT # 02-6100 was used as a standard for quantification.
- Isotyping of the anti-PTX3 antibody was performed using ImmunoPure Monoclonal Antibody Isotyping Kit II (PIERCE CA T # 37502), and the method was in accordance with the attached manual. As a result of isotyping, IgGl, IgG2a, and IgM class antibodies were obtained.
- PTX3 protein was prepared so as to be 100 gZ, and emulsified using FCA (Freund's complete adjuvant (H37 Ra), Difco (3113-60), betaton Dickinson (cat # 231131)) Was administered subcutaneously.
- FCA Full's complete adjuvant
- Difco (3113-60) Difco (3113-60)
- betaton Dickinson cat # 231131
- FIA Complete Freund's adjuvant, Difco (0639-60), Betaton Dickinson (cat # 263910).
- 100 gZ FIA emulsions were subcutaneously administered every two weeks, for a total of five immunizations.
- a sandwich ELISA system for PTX3 was constructed as follows.
- antibodies coated on 96-well plates include monoclonal antibodies such as PPMX0101 (FERM P-1 9697), PPMX0102, PPMX0112, and PPMX0148, and a polyclonal antibody labeled with biotin as an antibody to detect PTX3 bound thereto. (ALEXIS or the antibody prepared in Example 7) was used.
- the antibody was used for Piotin-dani using Sulfo-NHS-LC Biotin (CAT # 21335) manufactured by Pierce.
- Anti-III antibody was diluted with PBS (-) to a concentration of 10 ⁇ g ZmL in a 96-Elimno plate. This was coated and incubated at 4 ° C. The next day, after washing three times with 300 LZwell washing buffer (0.05% (v / v) Tween20, PBS), 150 ⁇ L of ABI immunoassay stabilizer (ABI # 10—601—001) was used. Calo, blocking was done. After several hours at room temperature or after storage at 4 ° C overnight, the purified protein, human serum, etc. are appropriately diluted with a dilution buffer (50 mM Tris-Cl pH 8.0, 0.15 M NaCl) containing animal serum.
- a dilution buffer 50 mM Tris-Cl pH 8.0, 0.15 M NaCl
- the ELISA standard curve was obtained using a concentration-tested PTX3 protein standard (ALEXIS), and was used for 30, 10, 3, 1, 0.3, 0.1, 0.03, 0. It was made. Further, a standard curve was obtained in the same manner as described above using the antibody of Non-patent Document 3, ie, the MNB4 antibody purchased from ALEX IS, for sensitivity comparison of ELISA. Comparison of the measurement system prepared this time with the measurement by the antibody of Non-Patent Document 3 (Fig. 3) confirmed that the sensitivity of the new measurement system was clearly superior.
- ALEXIS concentration-tested PTX3 protein standard
- Blood was collected before the start of statin administration, and then blood was collected after administration of atomatic pastatin once a day for 1 week after dinner.
- the collected blood sample was subjected to incubation at 37 ° C for 2 hours, followed by serum separation at 1000 rpm for 10 minutes. Collect serum supernatant components and Stored at ° C.
- the serum PTX3 protein concentration was measured using an ELISA system newly constructed using these sera. As a result, the serum PTX3 protein concentration was decreased in 6 of the 6 cases (Table 1). Further, as a result of the t-test, the PTX 3 concentration after one week of administration of atovatic pastatin was significantly (p 0.01) lower.
- serum and plasma have been used as blood samples for measuring blood components.
- the examination was performed using serum, which is the most common blood sampling method.
- the difference in blood PTX3 concentration between blood collection methods was examined in the addition test of standard PTX3 in the ELIS A system using the PPMX0112 monoclonal antibody.As shown in Table 2, the blood collection method using EDTA was optimal. Something became clear.
- Example 12 it was found that the preservability of PTX3 at 80 ° C could not be ensured, but it was speculated that the cause was not only deterioration of PTX3 but also changes in other plasma components.
- EDTA or a protease inhibitor to the buffer used for the measurement was examined using an ELISA system using the PPMX0112 monoclonal antibody. As a result, improvement of the measured value was confirmed by the addition of EDTA.
- various surfactants such as Tween20 and Bridje were added to the plasma sample in the presence of EDTA. As shown in Table 5, the improvement effect that was insufficient with EDTA alone could be obtained with Tween20.
- Example 6 Using the monoclonal antibody and the biotin-labeled polyclonal antibody prepared in Example 6, optimal antibody selection under the improved measurement conditions of Example 13 was performed. That is, the EDTA-collected plasma is diluted with a buffer containing EDTA and a detergent. Next, the diluted sample is added to a plate pre-coated with various monoclonal antibodies. After washing, a monoclonal antibody was selected by adding a biotin-labeled polyclonal antibody. As a result, since PPMX0102 was selected as the optimal antibody, the monoclonal antibody was designated as FERM AP-20395 by the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (Tsukuba Ito, Ibaraki Pref. — 1 Deposited to Chuo No. 6).
- Example 15 Addition of PTX3 to Plasma of Healthy Subjects by Conventional Measurement System and Newly Constructed Measurement System Recovery Test and Stability Test after Cryopreservation
- PTX3 addition concentration (PTX3ng / mL) (PTX3ng / mL) (PTX3ng / mL) (PTX3ng / mL)
- PTX3 concentration was measured by E-LISA using PPMX0102 (FERM AP-20395). That is, coronary artery (CA) normal coronary artery group (ANCA) 25 people, stable angina group (AP) 9 people, unstable angina group (UAP) 3 people, myocardial infarction group (AMI) 3 people PTX3 concentration in plasma was measured and compared between groups. As a result, PTX3 values were significantly increased in the UAP CA group compared to the ANCA CA group, and PTX3 values were significantly increased in the AMICA group compared to the ANCA CA and AP CA groups. As shown in FIG.
- CA coronary artery
- ANCA normal coronary artery group
- AP stable angina group
- UAP unstable angina group
- AMI myocardial infarction group
- CRP an inflammatory protein that belongs to the pentraxin family like PTX3 Used as a diagnostic. Unlike PRP hepatocytes, PTX3-producing cells are restricted to vascular constituent cells such as endothelial and smooth muscle cells.To demonstrate the vascular specificity of PTX3, the blood concentration in three cold patients was determined. It was measured. As a result, as shown in Table 8, the pre-onset value was 0.67—1.15 and the average was 0.92 ngZmL, while after onset 0.79—1.06 and the average was 0.90 ngZmL. The measured values were almost the same. These results confirmed that PT X3 is a highly specific marker for blood vessels, not a diagnostic marker for systemic inflammatory response, and is useful for determining the degree of vascular damage.
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WO2007055340A1 (ja) * | 2005-11-11 | 2007-05-18 | The University Of Tokyo | Ptx3高感度測定法 |
JP2007534951A (ja) * | 2004-04-29 | 2007-11-29 | ファーマ ディヴェロップメント ソシエタ ア レスポンサビリタ リミタータ | タンパク質ptx3測定用の単クローン抗体、ハイブリドーマ、改良方法、及び前記測定用キット |
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JP2010513917A (ja) * | 2006-12-22 | 2010-04-30 | ヒューマニタス・ミラソーレ・エス.ピー.エー. | ロングペントラキシンptx3の血漿レベルを測定するための方法 |
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US7955807B2 (en) | 2005-11-11 | 2011-06-07 | The University Of Tokyo | Method of measuring PTX3 with high sensitivity |
JP5137015B2 (ja) * | 2005-11-11 | 2013-02-06 | 国立大学法人 東京大学 | Ptx3高感度測定法 |
JP2009525463A (ja) * | 2005-12-29 | 2009-07-09 | ヒューマニタス・ミラソーレ・エス.ピー.エー. | 妊娠における炎症性内皮機能障害に対する診断試験 |
JP2010513917A (ja) * | 2006-12-22 | 2010-04-30 | ヒューマニタス・ミラソーレ・エス.ピー.エー. | ロングペントラキシンptx3の血漿レベルを測定するための方法 |
JP2012053034A (ja) * | 2010-08-06 | 2012-03-15 | Perseus Proteomics Inc | 炎症状態の検出方法 |
JP2015529335A (ja) * | 2012-09-12 | 2015-10-05 | バーグ エルエルシー | 心臓毒性剤の同定におけるマーカーの使用 |
WO2019056991A1 (zh) * | 2017-09-19 | 2019-03-28 | 臻崴生物科技有限公司 | 单克隆抗体或其抗原结合片段及其用途 |
WO2021260178A1 (en) | 2020-06-25 | 2021-12-30 | Humanitas Mirasole S.P.A. | Ptx3 as prognostic marker in covid-19 |
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JPWO2005080981A1 (ja) | 2007-10-25 |
EP1720014A4 (en) | 2008-01-16 |
JP4667372B2 (ja) | 2011-04-13 |
EP1720014A1 (en) | 2006-11-08 |
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