JP5488885B2 - Disease markers for infectious diseases - Google Patents

Description

  The present invention relates to a disease marker for infectious diseases, and relates to the field of diagnosis of infectious diseases such as sepsis using the disease markers.

  Infectious disease is a general term for diseases caused by infection with pathogens such as bacteria, fungi, viruses, parasites, and abnormal prions. It is called an infectious disease as a series of flows including those that come out. With regard to infectious diseases, infectious diseases that deal with diagnosis, treatment, and prevention of infectious diseases are being developed against the background of advances in microbiology, immunology, pharmacology, internal medicine, external science, and public health. Even so, infectious diseases still account for about one-fourth of the causes of death worldwide. In particular, malaria, tuberculosis, AIDS, and intestinal infections are major problems in developing countries. In developed countries, the spread of multidrug-resistant bacteria and patients with post-surgery and immunosuppressed conditions due to the development of advanced medicine The situation is not fully resolved, such as the increase in opportunistic infections.

  Infectious diseases occur in various organs in the body and cause inflammation mainly in the infected organs such as encephalitis, rhinitis, pharyngitis, pneumonia, infective endocarditis, hepatitis, enteritis and the like. A clinical syndrome in which the cause of infection is bacteria and the source of the fungus is phlebitis, endocarditis or lymphangitis is called sepsis. Sepsis is a systemic inflammatory response syndrome that, without treatment, can result in early death from shock, multiple organ failure, disseminated intravascular coagulation (DIC), and the like. Since sepsis often develops when the immunity in the body is reduced, the treatment results are not good.

  Diagnosis of sepsis includes examination of common inflammatory reactions such as white blood cell count and C-reactive protein (CRP) in serum, and searching for causative bacteria by blood culture. Although the identification of the causative bacteria is important in determining the treatment policy for sepsis, it takes several days to one week because the culture of the bacteria takes time. Therefore, until the causative bacterium is determined, an antibiotic that is considered to be highly sensitive from the original disease or disease state is administered, and after the causative bacterium is determined, an antibiotic that is highly sensitive to the bacterium is administered.

  In the diagnosis of inflammation, CRP is used as a typical diagnostic marker. However, the value of CRP increases when inflammation has already occurred. From the viewpoint of early diagnosis of infectious diseases, it cannot be said that CRP alone is perfect. Here, pentraxin 3 (also referred to as PTX3) is known to belong to the same protein family as CRP. Pentraxin 3 has been identified as a secreted protein that is expressed in human vascular endothelial cells stimulated with interleukin 1β (IL-1β) (Non-patent Document 1), including CRP, serum amyloid A, and the like. Is a protein belonging to the pentraxin family. The C-terminal side has a common domain peculiar to the pentraxin family, the N-terminal side has a domain structure peculiar to pentraxin 3, and forms an octamer as a whole via disulfide bonds (non- Patent Document 2).

  Pentraxin 3 itself has been found to increase in blood levels in patients with acute myocardial infarction, a type of inflammatory reaction, and to be detected in progressive arteriosclerotic plaque that can be an indicator of small vessel inflammation. It has been suggested to be involved in (non-patent documents 3 to 5). Therefore, it has already been reported that pentraxin 3 is useful as a marker for infection or inflammation (Patent Documents 1 to 3).

  The physiological role of pentraxin 3 is said to be involved in, for example, recognition or removal of pathogens that have invaded the body, or pregnancy (Non-Patent Document 6), but there are still many unclear points regarding their functions. Yes. Pentraxin 3 has also been reported to bind to other proteins (Non-patent Document 7), and is thought to function in cooperation with the other proteins. It is not.

International Publication No. 2005/080981 Pamphlet International Publication No. 2005/106494 Pamphlet International Publication No. 2007/055340 Pamphlet

Breviario, F et al., J. Biol. Chem. 267: 22190-22197, 1992. Inforzato, A et al., J. Biol. Chem. 283: 10147-10161, 2008. Fazzini, F et al., Arthritis Rheum. 44: 2841-2850, 2001. Peri, G et al., Circulation. 102: 636-641, 2000. Rolph, MS et al., Arterioscler Thromb Vasc Biol. 22: e10-4, 2002. Garlanda, C et al., Nature 420: 182-186, 2002. Mantovani, A et al., J. Clin. Immunol. 28: 1-13, 2008.

  Although it is possible to diagnose infectious diseases in a wide range by examining only pentraxin 3, it is necessary to clarify the physiological role of pentraxin 3 itself in order to increase the accuracy of the diagnosis. Further ingenuity is required in the method. Therefore, the problem to be solved by the present invention is to provide a tool capable of quickly and early diagnosis of infectious diseases, particularly sepsis.

  The present inventors have focused on pentraxin 3 in which the blood concentration in patients with infectious diseases is extremely higher than that in healthy individuals, and examined molecules that can be used as disease markers for infectious diseases through functional analysis. . As a result of extensive research, the present inventors have found that a complex of azulocidin and pentraxin 3 is detected in the blood of a septic patient, and that azulocidin and pentraxin 3 can actually form a complex. As a result, the present invention was completed.

That is, the present invention is as follows.
(1) A disease marker for infectious diseases comprising pentraxin 3 and azulocidin.
(2) The disease marker according to (1), wherein pentraxin 3 and azulocidin form a complex.
(3) The disease marker according to (1) or (2), wherein the infectious disease is sepsis.
(4) A method for detecting the presence or absence of an infectious disease, comprising the following steps (A) to (E):
(A) a step of contacting a sample collected from a test animal with magnetic particles bound with an anti-pentraxin 3 antibody;
(B) recovering the magnetic particles;
(C) separating the component derived from the sample captured by the anti-pentraxin 3 antibody from the magnetic particles recovered in step (B), and concentrating the component;
(D) A step of examining the presence or amount of pentraxin 3 and azulocidin in the component concentrated in step (C),
(E) A step of detecting the presence or absence of an infectious disease based on the result of the step (D).
(5) The detection method according to (4), wherein the sample collected from the test animal in step (A) is heparin-treated or ethylenediaminetetraacetic acid-treated plasma, serum, or a combination thereof.
(6) The detection method according to (4) or (5), wherein the infectious disease is sepsis.
(7) The detection method according to any one of (4) to (6), wherein the magnetic particles in step (A) are coated with protein G.
(8) The detection method according to any one of (4) to (7), further including a step (C ′) after the step (C):
(C ′) A step of decomposing the component concentrated in step (C).
(9) The detection method according to (8), wherein the degradation in step (C ′) is trypsin digestion.
(10) The determination of whether or not there is an infection in step (E) is obtained by comparing the result of step (D) obtained for the test animal with the result of step (D) obtained for the normal control, The detection method according to any one of (4) to (9), wherein the presence of azulocidin or the increase in the amount thereof is used as an index.
(11) A method for simultaneously detecting the presence or absence of an infectious disease and the pathogen of the infectious disease, including the following steps (a) to (g):
(A) contacting a sample collected from a test animal with magnetic particles bound with an anti-pentraxin 3 antibody;
(B) recovering the magnetic particles;
(C) separating the component derived from the sample captured by the anti-pentraxin 3 antibody from the magnetic particles recovered in step (b), and concentrating the component;
(D) a step of performing mass spectrometry of the component concentrated in step (c),
(E) identifying a molecule that binds to pentraxin 3 based on the analysis result of step (d),
(F) Based on the identification result of step (e), a step of examining the presence or amount of pentraxin 3 and azulocidin, and a bacterial or fungal-derived molecule,
(G) A step of detecting presence or absence of an infection and pathogenic bacteria of the infection based on the result of the step (f).
(12) The detection method according to (11), wherein the sample collected from the test animal in step (a) is heparin-treated or ethylenediaminetetraacetic acid-treated plasma, serum, or a combination thereof.
(13) The detection method according to (11) or (12), wherein the infectious disease is sepsis.
(14) The detection method according to any one of (11) to (13), wherein the magnetic particles in step (a) are coated with protein G.
(15) The detection method according to any one of (11) to (14), further including a step (c ′) after the step (c):
(C ′) A step of decomposing the component concentrated in step (c).
(16) The detection method according to (15), wherein the degradation in step (c ′) is trypsin digestion.
(17) The presence or absence of infection in step (g) and the determination of the pathogen of the infection are compared with the result of step (f) obtained for the normal control in the result of step (f) obtained for the test animal. The detection method according to any one of (11) to (16), wherein pentraxin 3 and azulocidin and bacteria or fungi-derived molecules are present or the amount thereof is increased.
(18) A kit for diagnosing infectious diseases, comprising a magnetic particle to which an anti-pentraxin 3 antibody is bound, and a substance capable of detecting pentraxin 3 and azulocidin.
(19) The diagnostic kit according to (18), wherein the magnetic particles are coated with protein G.
(20) The substance capable of detecting pentraxin 3 and azulocidin is selected from the group consisting of an internal standard substance that can be used for mass spectrometry of pentraxin 3 and azulocidin, or an antibody, a ligand, and a receptor that recognize pentraxin 3 and azulocidin The diagnostic kit according to (18) or (19), which is one or more molecules.

  If the disease marker for infectious diseases of the present invention is used, a more accurate diagnosis of infectious diseases can be performed, and considering that the complex formed by pentraxin 3 can induce the immunity of animals, the present invention It is also possible to perform early diagnosis. In addition, if the detection method of the present invention using the disease marker is used, it is possible to efficiently inspect or diagnose an infectious disease, and in order to detect the causative bacterium of the infectious disease, the culture of the bacterium is not particularly required. Therefore, it is possible to provide a rapid detection method in a short time, and it can be used for labor-saving inspection or diagnosis of infectious diseases. Furthermore, when plasma or serum is used as a test sample in the detection method of the present invention, a small amount of pentraxin 3 complex can be purified from a blood component having a dynamic range of 10 digits with high efficiency. Markers can be detected comprehensively and simultaneously, and proteomic analysis can be applied in the diagnosis of diseases from blood samples.

It is the figure which showed the result of the electrophoresis of the pentraxin 3 complex in the plasma extract | collected from the test subject. A. It is the figure which isolate | separated the protein which isolate | separated the pentraxin 3 plasma in the plasma extract | collected from the test subject, and investigated the isolate | separated protein by SDS-PAGE. Lane PPMX0103 shows the results using the anti-pentraxin 3 antibody, and Lane Mouse IgG shows the results using the control anti-mouse IgG antibody. An asterisk band indicates pentraxin 3, and a circle indicates a pentraxin 3 binding protein band. The numerical value at the left end indicates the molecular weight (kDa) of the protein. B. It is the figure which showed the result of having immuno-isolated the pentraxin 3 complex in the plasma extract | collected from the test subject, and having investigated the isolate | separated protein by Western blotting. As controls, 6 ng of recombinant pentraxin 3 (PTX3) and 5% of the plasma used for immunoseparation (5% input) were used. The values shown below the various bands are the concentrations of the recombinant PTX3 bands. Represents the amount of pentraxin 3 calculated from It is a figure which shows the result of the binding assay of pentraxin 3 and azulocidin or C1q. A. It is the figure which showed the result of the binding analysis by the binding assay of pentraxin 3 and azulocidin. B. It is the figure which showed the result of the binding analysis by the binding assay of pentraxin 3 and C1q. Black squares indicate the results of analysis with addition of CaCl ₂ (4 mM), and white squares indicate the results of analysis with addition of EDTA (4 mM). In each graph, the horizontal axis represents the concentration of pentraxin 3 (PTX3), and the vertical axis represents the absorbance at a wavelength of 450 nm. It is the figure which showed the result of the binding analysis of pentraxin 3 and azulocidin by BIAcore. The horizontal axis of the graph indicates time (seconds), and the vertical axis indicates the amount of binding between pentraxin 3 and azulocidin, which is analyzed by adding CaCl ₂ (2 mM) or EDTA (3 mM).

1. Disease marker for infectious diseases The present invention provides a disease marker for infectious diseases comprising pentraxin 3 and azulocidin.

  Pentraxin 3 (hereinafter also referred to as PTX3) in the present invention is a protein belonging to the pentraxin family, and is characterized by being directly produced in a short time from vascular endothelial cells, vascular smooth muscle, macrophages, neutrophils, and the like. doing.

  In the present invention, PTX3 is a protein derived from any mammal. Examples of mammals include humans and mammals other than humans. Examples of mammals other than humans include rodents such as mice, rats, hamsters, and guinea pigs, and laboratory animals such as rabbits, pigs, cows, and goats. , Primates such as domestic animals such as horses and sheep, pets such as dogs and cats, monkeys, orangutans and chimpanzees. In order to use for examination or diagnosis of human infectious diseases, PTX3 derived from human is preferable.

  Human-derived PTX3 is a protein consisting of a single-chain polypeptide having a total length of 381 amino acids, and the amino acid sequence and base sequence thereof are registered in GenBank as Accession No. BC039733. In the amino acid sequence, the 1st to 17th portions from the N-terminus are signal peptides, which are cleaved in the process of being secreted outside the cells to become mature proteins. The N-terminal site having a domain structure peculiar to PTX3 is the 18th to 178th amino acid sequence portion from the N-terminus, and the C-terminal site highly homologous to CRP and serum amyloid A is 179th to 381st from the N-terminus. Corresponds to the amino acid sequence portion of.

  PTX3 in the present invention includes not only a polypeptide represented by a specific amino acid sequence obtained from any mammal as described above, but also mutants thereof as long as their biological functions are equivalent. Derivatives, mature forms, amino acid modifications and splice variants are included. Here, the mutants include naturally occurring allelic mutants and mutants having an amino acid sequence modified by artificial substitution, deletion, addition and insertion of amino acids. Examples of the mutant include those having at least 70%, preferably 80%, more preferably 95%, and even more preferably 97% homology with a polypeptide having no mutation. Specific examples of amino acid modifications include phosphorylated, hydroxylated, and methylated amino acids.

  In addition, azulocidin in the present invention is a protein belonging to the serine protease family, but the amino acid residue in the active site has been mutated and has no enzyme activity itself. Azulocidin is a cationic antibacterial protein having bactericidal activity against gram-negative bacteria, and has the characteristic of increasing vascular permeability in the vascular endothelium when inflammation occurs due to the presence in neutrophils and the like. Azulocidin is also called azulocidin 1 as its name.

  Azurosidin in the present invention is also a protein derived from any mammal like PTX3. Examples of mammals include humans and mammals other than humans. Examples of mammals other than humans include mice, rats, Examples include rodents such as hamsters and guinea pigs, laboratory animals such as rabbits, domestic animals such as pigs, cows, goats, horses and sheep, pets such as dogs and cats, and primates such as monkeys, orangutans and chimpanzees. In order to use for examination or diagnosis of human infectious diseases, it is preferable to use human-derived azulocidin.

  Human-derived azurocidin is a protein consisting of a single-chain polypeptide having a total length of 251 amino acids, and the amino acid sequence and base sequence thereof are registered in GenBank as Accession No. M96326. In the amino acid sequence, the 1st to 26th portion from the N-terminal is presumed to be a signal peptide, and it is considered that it is cleaved together with the 3 amino acid residues at the C-terminal to become a mature protein.

  In the case of azulocidin in the present invention, not only a polypeptide represented by a specific amino acid sequence obtained from any mammal as described above as in the case of PTX3, but also a biological function equivalent thereto, Such variants, derivatives, matures, amino acid modifications and splice variants are included. Here, the mutants include naturally occurring allelic mutants and mutants having an amino acid sequence modified by artificial substitution, deletion, addition and insertion of amino acids. Examples of the mutant include those having at least 70%, preferably 80%, more preferably 95%, and even more preferably 97% homology with a polypeptide having no mutation. Specific examples of amino acid modifications include phosphorylated, hydroxylated, and methylated amino acids.

  In the present invention, the term “disease marker” refers to screening for candidate substances useful for diagnosing the presence or absence, degree of morbidity, presence or absence of improvement, and degree of improvement of infectious diseases, and for the prevention and treatment of infectious diseases. Therefore, it is used directly or indirectly. The disease marker is characterized in that it contains PTX3 and azulocidin, but in the present invention, the disease marker is not limited to being a protein, and it means that the gene encoding the protein is also included. To do. In order to detect the gene and protein expressed in vivo, tissue or cell based on this property, because the expression of the gene and protein varies in vivo in relation to the infection. These probes, primers or antibodies can be used effectively.

  Further, the disease marker of the present invention is not particularly limited, and further, Filaggrin-2, Fibrinogen beta chain, Mannan-binding lectin serine protease 1, Protein AMBP, Vitronectin, Clusterin, Lipopolysaccharide-binding protein, Alpha-2-macroglobulin, Histone H3.1, Ig kappa chain V-III region WOL, Myeloperoxidase, Versican core protein, Haptoglobin, Histone H2B type 1-B, Platelet factor 4, Alpha-1-antitrypsin, Serum amyloid A protein, Thrombospondin-1, Ceruloplasmin, Prothrombin, Coagulation factor V, Collectin-11, Alpha-1-antichymotrypsin, Hemoglobin subunit beta, Apolipoprotein AI, Hemoglobin subunit alpha, Mannan-binding lectin serine protease 2, Alpha-2-HS-glycoprotein, Antithrombin-III, Hemoglobin subunit delta , Serum amyloid P-component, Vitamin K-dependent protein S, Coagulation factor XI, Apolipoprotein E, Histone H2A type 1-D, Phosphatidylinositol-5-phosphate 4-kinase type-2 alpha, Coagulation factor X, Multimerin-1, Serum depriv Other proteins such as ation-response protein, tissue factor pathway inhibitor, and secreted phosphoprotein 24 can be included. PTX3 and azulocidin can be used in combination with one or more of the above proteins. As the protein to be combined, it is preferable to use Clusterin, Lipopolysaccharide-binding protein, Myeloperoxidase, Alpha-1-antitrypsin or Alpha-2-HS-glycoprotein. The amino acid sequence and base sequence of the protein are known and registered on the NCBI website and the like. In addition, Tables 1 to 4 described below describe the Accession Number of the amino acid sequence.

  In addition to the above, the disease marker of the present invention may further contain a protein component derived from the causative bacterium of the infectious disease. The protein component derived from the causative bacterium can be used alone or in combination of PTX3, azulocidin and the other proteins described above. The protein component derived from the causative bacterium is not particularly limited. For example, oligopeptides having 5 to 50 amino acid residues are exemplified, and examples thereof are described in Tables 5 to 7 described later.

  Moreover, in the disease marker of the present invention, PTX3 and azulocidin may form a complex. In addition to PTX3 and azulocidin, when one or more proteins are included, and when protein components derived from causative bacteria are further included, these proteins may also form a complex with PTX3. Here, the complex refers to a state in which PTX3 is bound to an anti-PTX3 antibody in a state where it can be immunoseparated when contacted with the anti-PTX3 antibody in a solution. PTX3 and azulocidin may be directly bound, or may be indirectly bound via another molecule or the like. Examples of the bonding mode include intermolecular hydrogen bonding, covalent bonding, van der Waals bonding, electrostatic interaction, etc., but PTX3, azurocidin, and other proteins are maintained in a state where they are not dissociated from each other. If it is a thing, the form in particular will not be restrict | limited.

  The disease marker of the present invention may be present in a biological sample derived from a test animal, but is preferably isolated from the sample or concentrated in a predetermined solution after isolation. Alternatively, PTX3, azulocidin, the above protein and the protein component derived from the causative bacterium may be produced or synthesized in vitro, and these proteins may be stored in separate containers or may be stored together in one container. . The disease marker of the present invention may contain other components as long as the protein or the like is the main component. Examples of other components include, but are not limited to, solvents such as buffer solutions and physiological saline, stabilizers, bacteriostatic agents, preservatives, and the like. The main component protein and peptide can be produced or synthesized by a method known per se. Further, the disease marker of the present invention may be a combination of numerical values of the amount of protein and peptide contained in the marker, that is, a database. By creating a database of disease markers, various states of infectious diseases can be easily and quantitatively known from the measurement values obtained by the test method of the present invention.

  In the present invention, infectious diseases include various diseases caused by infection with pathogens such as bacteria, fungi, viruses, parasites, and abnormal prions. Sepsis refers to a pathological condition in which an intense inflammatory reaction is induced throughout the body by the action of an infected microorganism and its toxin beyond the infected area. Sepsis has a mortality rate of around 20%, and there is a demand for an early diagnosis method in addition to a pathogen identification method capable of selecting an appropriate treatment. In the present invention, it is preferable to select sepsis among the infectious diseases as a study case from the viewpoint that a phenomenon in which the blood concentration of PTX3 is particularly increased is observed.

2. The detection method of the presence or absence of the infection disease This invention also provides the detection method of the presence or absence of the infection disease including the following process (A)-(E).
(A) a step of contacting a sample collected from a test animal with magnetic particles bound with an anti-pentraxin 3 antibody;
(B) recovering the magnetic particles;
(C) separating the component derived from the sample captured by the anti-pentraxin 3 antibody from the magnetic particles recovered in step (B), and concentrating the component;
(D) A step of examining the presence or amount of pentraxin 3 and azulocidin in the component concentrated in step (C),
(E) A step of detecting the presence or absence of an infectious disease based on the result of the step (D).

  The test animal in the present invention is not particularly limited, and can be any animal. Examples of the animal include humans and mammals other than humans. Examples of mammals other than humans include rodents such as mice, rats, hamsters, and guinea pigs, and laboratory animals such as rabbits, pigs, cows, and goats. , Primates such as domestic animals such as horses and sheep, pets such as dogs and cats, monkeys, orangutans and chimpanzees.

  A biological sample derived from a test animal as a sample is not particularly limited as long as it may contain PTX3 and azulocidin, but a sample collected from a living body such as a mammal is preferable, and more preferably collected from a human. Sample. Specific examples of the biological sample include blood, plasma, serum, extravascular fluid, interstitial fluid, cerebrospinal fluid, synovial fluid, pleural fluid, lymph fluid, saliva, semen, tears, urine and the like. Can do. Among these, the sample of the present invention is preferably blood, serum, plasma, saliva, semen, tears, urine, more preferably blood, serum, plasma, more preferably, from the viewpoint of less invasion to animals. Plasma or serum.

  When plasma or serum is used, it can be prepared by collecting blood from a test animal according to a conventional method and separating the liquid component. According to the present invention, depending on the physical properties of PTX3 and azurocidin to be detected, contaminants can be separated and removed from the fractions containing these proteins in advance using filter filtration or various columns. When using plasma, heparin, ethylenediaminetetraacetic acid (EDTA), antithrombin III, protein C, protein S, aspirin, etc. are used as anticoagulants to suppress coagulation due to fibrinogen, fibrin, thrombin, etc. Can be processed. In the present invention, heparin or EDTA is preferably used from the viewpoint of handleability. In addition, when EDTA is used, EDTA chelates divalent metal ions such as calcium ions, and therefore heparin is more used from the viewpoint of not affecting divalent metal ions in plasma. preferable.

  In the present invention, the presence or absence of PTX3 and azulocidin or their amounts can be examined according to the type of biological sample collected from the test animal (eg, plasma, serum, blood, etc.). By using the detection method of the present invention in combination with PTX3 and azurocidin that are detected in serum, PTX3 and azulocidin and the like detected in serum, it is possible to diagnose infection with higher accuracy. In the plasma, as described above, a result corresponding to the sample treated with heparin, EDTA, or the like can be obtained and combined to be used for a method for detecting the presence or absence of infection. .

  The anti-pentraxin 3 antibody in the present invention is not particularly limited as long as it can recognize PTX3, and may be a monoclonal antibody or a polyclonal antibody. Of these, the anti-PTX3 antibody is preferably a monoclonal antibody from the viewpoint of recognizing a specific epitope and being stably produced.

  The origin of the anti-PTX3 antibody is not particularly limited, and a mouse antibody, a rat antibody, a human antibody, a chimeric antibody prepared based on the gene encoding the monoclonal antibody, a single chain antibody, a humanized antibody, or the like can be used. it can. The form of the anti-PTX3 antibody is not particularly limited, and may be a low molecular weight antibody such as an antibody fragment (fragment), a modified antibody, or the like as long as the characteristic of recognizing PTX3 is not lost. Specific examples of the antibody fragment include Fab, Fab ', F (ab') 2, Fv, Diabody and the like.

  In the present invention, examples of the anti-PTX3 antibody include 16B5 (Bottazzi, B et al., J. Biol. Chem. 272: 32817-32823, 1997.), 1C8 (Bottazzi B et al., J. Biol. Chem). 272: 32817-32823, 1997.), MNB4 (Peri, G et al., Circulation. 102: 636-641, 2000.), MNB6 (Peri, G et al., Circulation. 102: 636-641, 2000) .), MNB10 (Accession Number ABC PD04001), Pen-3 (Accession Number ABC PD01004), PPMX0101 (Accession Number FERM P-19697), PPMX0102 (Accession Number FERM BP-10326), PPMX0103, PPMX0112, PPMX0148, etc. An antibody is mentioned.

  The anti-PTX3 antibody used in the present invention may specifically bind to PTX3, but preferably recognizes a three-dimensional epitope of PTX3. More preferred is an antibody that exhibits high binding affinity for the three-dimensional structure of PTX3 and does not cross-react with CRP or serum amyloid A. Here, the three-dimensional structure epitope refers to an epitope that recognizes the secondary structure, tertiary structure, or quaternary structure of full-length PTX3, excluding the epitope that recognizes the primary structure among the epitopes that recognize the full-length PTX3 portion. Here, it is known that the three-dimensional structure of a protein has a hierarchical structure of a primary structure, a secondary structure, a tertiary structure, and a quaternary structure (Nanyamado Medical Dictionary Revision 17th Edition (1990)). The preferred antibody of the present invention recognizes non-reduced PTX3, does not react with the PTX3 fragment, but recognizes the three-dimensional structural epitope of PTX3. Therefore, by reacting strongly with intact PTX3, PTX3 that retains higher order structures in the body can be identified. The anti-PTX3 antibody used in the present invention can be produced according to the method described in WO2005 / 106494, WO2005 / 080981, and WO2007 / 055340.

The magnetic particles used in the present invention are magnetic particles, which can be dispersed or suspended in an aqueous medium and can be separated from the dispersion or suspension by applying a magnetic field. Any particles can be used if present. The kind of the particle | grains is not specifically limited, The particle | grains comprised by the combination of organic particle | grains or inorganic particle | grains (a metal particle is included), or organic and inorganic are contained. The magnetic particles in the present invention are preferably those containing a magnetic substance inside the organic (polymer) particles, and further, this magnetic substance is contained only inside the particles and is not exposed to the particle surface. It is more preferable. Examples of such a magnetic material include metals such as iron, cobalt, manganese, chromium or nickel, alloys of the metals, or salts, oxides, borides or sulfides of the metals; rare earths having high magnetic susceptibility. And the like (eg, hematite or ferrite). Specific examples of the magnetic material include, for example, ferromagnetic rules such as magnetite (iron trioxide (Fe ₃ O ₄ )), γ-heavy ferric oxide (γ-Fe ₂ O ₃ ), FePd, FePt, and CoPt. Alloys can also be used. A preferable magnetic material in the present invention is a metal oxide, and particularly, a material selected from the group consisting of iron oxide and ferrite (Fe, M) ₃ O ₄ . Here, iron oxide includes, among others, magnetite, maghemite, or a mixture thereof. Wherein M is a metal ion that can be used with the iron ion to form a magnetic metal oxide, typically selected from transition metals, and most preferably Zn ²⁺ , Co ²⁺ , Mn ²⁺ , Cu ²⁺ , Ni ²⁺ , Mg ^{2+ and the} like, and the molar ratio of M / Fe is determined according to the stoichiometric composition of the selected ferrite. The metal salt is supplied in solid form or in solution, but is preferably a chloride salt, bromide salt or sulfate. Among these, iron oxide and ferrite are preferable from the viewpoint of safety, and magnetite (Fe ₃ O ₄ ) is particularly preferable.

  The size of the magnetic particles in the present invention is not particularly limited, and may be any of nanoparticles, microparticles, and milliparticles, but is preferably nanoparticles or microparticles. Among these, the magnetic particles in the present invention are preferably particles having an average particle diameter of 50 nm to 20 μm, more preferably particles having an average particle diameter of 100 nm to 10 μm, and still more preferably microparticles having an average particle diameter of 1 to 5 μm. . Moreover, the average particle diameter of the magnetic substance contained in the magnetic particles is preferably 0.1 to 10 nm, more preferably 0.5 to 5 nm, and further preferably 1 to 3 nm. If the average particle diameter of the magnetic material exceeds 10 nm, the effect of residual magnetization appears, and even after the magnetic field is removed, the mutual coupling between the magnetic particles remains, which affects the particle dispersibility. In addition, the particle shape of the magnetic particle and the magnetic substance included in the magnetic particle in the present invention is not necessarily a perfect spherical shape, and may be a deformed particle such as a spheroid. In that case, the average particle diameter of the particles that are not perfectly spherical can be calculated by taking the average value of the longest diameter and the shortest diameter of the particles. The average particle diameter can be measured by a method known per se, for example, by a light scattering method.

  The content of the magnetic material is 1 to 99% by weight, and particularly preferably 20 to 70% by weight, based on the entire magnetic particles in the present invention. If the content of the magnetic substance is too small, a good magnetic force cannot be obtained in the magnetic particles themselves, and in the detection method of the present invention, it takes a considerably long time to recover the magnetic particles brought into contact with the sample collected from the test animal. Is not preferable because high time efficiency may not be obtained.

The magnetic particles in the present invention can be prepared by the following methods (i) to (iv).
(I) Polymerization is performed by mixing a magnetic substance with a polymerization component containing a monomer constituting a polymer. As a polymerization method, an ordinary method for obtaining a polymer aqueous dispersion can be used, and examples thereof include emulsion polymerization, suspension polymerization, and dispersion polymerization.
(Ii) A polymer component containing a monomer constituting the polymer is polymerized by a method of obtaining a normal polymer aqueous dispersion to obtain a polymer aqueous dispersion. A magnetic layer is formed on the particle surface.
(Iii) The polymer is dissolved in a solvent which is substantially insoluble in water and has a boiling point lower than that of water, and the magnetic substance is dispersed in this solution. This is dispersed in water using an appropriate emulsifier or dispersant, and the solvent is removed by distillation.
(Iv) A monomer is added and polymerized using the polymer aqueous dispersion obtained in (i), (ii) or (iii) above as a seed to form a polymer layer on the particle surface.

  In the present invention, the anti-PTX3 antibody and the magnetic particle are bonded to each other. Here, the binding refers to keeping the anti-PTX3 antibody and the magnetic particles in contact with each other or maintaining a certain distance and not dissociating from each other. Further, the anti-PTX3 antibody and the magnetic particles may be directly bonded to each other, or may be indirectly bonded through other molecules or the like. Examples of the method for binding the anti-PTX3 antibody and the magnetic particles include hydrophobic bond, ionic bond, chemical bond, affinity bond and the like, and chemical bond is more preferable. In addition, the method for binding the anti-PTX3 antibody to the magnetic particles is not particularly limited, and the antibody can be bound by a method known per se. For example, the method shown in Examples described later can be used.

  In addition, the surface of the magnetic particles in the present invention can be coated with an antibody binding substance such as protein G, protein A, protein L or the like. Among these, for example, when the magnetic particle surface is coated with protein G, the protein G-bound antibody is present at a certain distance from the particle surface, and the antigen-antibody reaction can be made highly sensitive. Furthermore, non-specific reactions can be reduced. Therefore, in the present invention, it is particularly preferable to use protein G. Moreover, although these coating methods are not specifically limited, For example, a hydrophobic bond, an ionic bond, a chemical bond, an affinity bond etc. are mentioned, A chemical bond is more preferable. The coating of the antibody binding substance or the like on the magnetic particles can be performed by a method known per se, and the magnetic particles coated with the antibody binding substance or the like are commercially available from JSR, Thermo Fisher SCIENTIFIC, GenScript, etc. It can be obtained by purchasing the product.

  In the step (A) of the present invention, the method of contacting the sample collected from the test animal and the magnetic particles bound with the anti-PTX3 antibody is not particularly limited as long as both are in contact with each other. Examples include a method in which the sample and anti-PTX3 antibody-bound magnetic particles are added to the solution and both are reacted. At this time, one of the sample and the anti-PTX3 antibody-binding magnetic particles can be added first, followed by the other, or the order can be reversed, or both can be added simultaneously. In addition, when both are added to the solution, the condition for bringing them into contact with each other is not particularly limited as long as the PTX3 complex that can exist in the sample and the anti-PTX3 antibody can bind to each other. It can be 5 minutes to 48 hours at ˜42 ° C., more preferably 30 minutes to 2 hours at 2 to 37 ° C. Examples of the solution include a buffer solution. Examples of the buffer solution include a phosphate buffer solution, an acetate buffer solution, a citrate buffer solution, a Tris-HCl buffer solution, a phosphate-buffered physiological saline, and a Tris solution. Examples include buffered saline.

  The method for collecting the anti-PTX3 antibody-bound magnetic particles in the step (B) of the present invention is not particularly limited. For example, the anti-PTX3 antibody-bound magnetic particles dispersed in the solution are collected by binding to a magnet. Such a method is mentioned. At this time, if the magnet is brought close to the outside of the container containing the solution containing the anti-PTX3 antibody-binding magnetic particles, the magnet and the magnetic particles can be easily detached after the magnetic particles are collected, which is a preferable mode. In the present invention, when anti-PTX3 antibody-bound magnetic particles are collected using a magnet, there is no particular limitation as long as at least half of the dispersed magnetic particles can be bound to the magnet. At 42 ° C., it can be 30 seconds to 2 hours, more preferably at 2 to 37 ° C., for 1 minute to 3 minutes. In addition, although it does not specifically limit as a magnet, For example, an alnico magnet, KS steel, MK steel, new KS steel, a ferrite magnet, a samarium cobalt magnet, a neodymium magnet etc. can be used, These are 1 type. Alternatively, two or more kinds can be used in combination.

  In the step (C) of the present invention, the sample-derived component captured by the anti-PTX3 antibody is separated from the magnetic particles recovered in the step (B), and the sample-derived component is concentrated. The method for separating the sample-derived component is not particularly limited as long as the anti-PTX3 antibody or the PTX3 complex that can be present in the sample can be separated from the magnetic particles. For example, surfactants, chaotropic ions The separation can be performed using a strong acid, a strong base, a modifier, a nonpolar solvent, a reducing agent, or the like. Examples of the surfactant used include NP-40, Tween 20, Tween 80, Triton X-100, and RapiGest. Chaotropic ions include perchlorate ion, thiocyanate ion, iodide ion, bromide. Ions, trichloroacetate ion and trifluoroacetate ion, urea, guanidine, etc., as strong acid or strong base, 0.1M glycine-HCl (pH 2.5), 1M acetic acid, 0.05% (w / v) trifluoro Acetic acid etc., urea, guanidine hydrochloride, dimethyl sulfoxide etc. as denaturing agents, ethylene glycol, dioxane etc. as nonpolar solvents, dithiothreitol (DTT), 2-mercaptoethanol as reducing agents (2-ME). Among these, it is preferable to use a surfactant from the viewpoint of handleability and ease of separation. Specifically, the magnetic particles recovered in the step (B) with a buffer solution containing a surfactant are 1 to If it is washed about 5 times, it can be separated efficiently. Examples of the buffer solution include phosphate buffer solution, acetate buffer solution, citrate buffer solution, Tris hydrochloride buffer solution, phosphate buffered saline solution, Tris buffered saline solution, ammonium bicarbonate buffer solution and the like. The concentration of the surfactant in the liquid can be 0.01 to 1% (w / v).

  The method for concentrating the sample-derived component is not particularly limited as long as the protein contained in the sample can be concentrated. For example, a trichloroacetic acid (TCA) precipitation method, an ultrafiltration concentration method, an ammonium sulfate precipitation method ( Salting out), organic solvent (eg, acetone, ethanol, propanol, methanol, etc.) precipitation method, water-soluble polymer (eg, polyethylene glycol, dextran etc.) precipitation method, acidic precipitation method, isoelectric point precipitation method and the like. Among these, in the present invention, the TCA precipitation method is particularly preferable from the viewpoint of removing surfactants, salts, and the like that affect mass spectrometry. Specifically, the sample-derived components are efficiently concentrated by adding a 10-30% (w / v) TCA solution, then adding acetone, cooling, and then washing with diethyl ether. Can do.

  In the present invention, the step (C ') may further include a step of decomposing the component concentrated in the step (C) after the step (C). A method for performing this degradation is not particularly limited as long as PTX3 and azulocidin can be detected. For example, trypsin digestion, lysyl endopeptidase, endopeptidase Asp-N, V8 protease, arginyl endopeptidase, proline Examples include degradation by protease such as specific endopeptidase, chemical degradation by bromocyanate, formic acid and the like. Among these, trypsin has an action of specifically cleaving the peptide bond on the C-terminal side of arginine and lysine in the protein, and can obtain a relatively physicochemically uniform peptide fragment. When using the protein mass spectrometry described later, trypsin digestion is particularly preferable. When trypsin digestion is performed in the present invention, for example, a final concentration of 0.2 to 5.0 ng / min is applied directly to the concentrated component derived from the sample or after the sample derived component is resuspended in a buffer solution or the like. μL trypsin can be added and allowed to react at 30-37 ° C. for 1-16 hours. In addition, when the step (C ′) is used, the step (D) can be a step of examining the presence or amount of pentraxin 3 and azulocidin in the component decomposed in the step (C ′).

  Although it does not specifically limit as a method of investigating the presence or absence or the amount of PTX3 and azulocidin in the sample-derived component in the step (D) of the present invention, for example, protein mass spectrometry can be used. Immunological methods can also be used. The details of the protein mass spectrometry are as described later. Examples of immunological methods include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay, luminescence immunoassay, immunoprecipitation method, immunoturbidimetric method, Western blot, immunostaining, immunodiffusion method, and the like. it can. Among these, an enzyme immunoassay is preferable, and an enzyme-linked immunosorbent assay (ELISA) (eg, sandwich ELISA) is particularly preferable.

  As a specific method of sandwich ELISA, when the presence or absence or amount of PTX3 is examined, for example, an anti-PTX3 antibody is immobilized on a support, and the concentrated sample-derived component (test sample) is added thereto to add anti-PTX3. The antibody is contacted with PTX3 (or a complex thereof) that may be present in the test sample, and incubation is performed to bind the anti-PTX3 antibody and PTX3 or the complex thereof, followed by washing to remove the labeled anti-PTX3 antibody. In addition, PTX3 or a complex thereof in a test sample can be detected by binding to PTX3 or a complex thereof remaining on the support and detecting the label. Examples of the support include synthetic resins such as silicone resin, polystyrene resin, polyacrylamide resin, nylon resin, and polycarbonate resin, and supports such as glass. These supports can be used in the form of a plate or the like. For example, a multiwell plate (96-well multiwell plate or the like) can be used. The peptide of the present invention can be bonded to the support by a commonly used method such as chemical bonding or physical adsorption. Any of these supports may be commercially available. Before adding the test sample, after washing the support, a blocking treatment with, for example, bovine serum albumin (BSA), gelatin, albumin, etc. is performed in order to prevent binding to a non-specific support such as protein. You can also When the blocking treatment is performed, the test sample may be added after washing again.

  The binding between the anti-PTX3 antibody and PTX3 or a complex thereof is usually performed in a buffer solution. As the buffer solution, for example, phosphate buffer solution, Tris buffer solution, citrate buffer solution, borate buffer solution, carbonate buffer solution and the like are used. Incubation conditions are not particularly limited as long as they are usually used. For example, incubation is performed at 4 to 40 ° C. for 10 minutes to 24 hours. The washing after incubation is not particularly limited as long as it does not interfere with the binding between the anti-PTX3 antibody and PTX3 or a complex thereof, and includes, for example, a surfactant such as Tween 20, Tween 80, Triton X-100, etc. A buffer solution or the like is used.

After washing, a labeled anti-PTX3 antibody (anti-PTX3 antibody labeled with a labeling substance) is added. As the label, a substance based on a known technique can be used. For example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, a hapten, or the like is used. As the radioisotope, for example, ³² P, ³³ P, ³⁵ S, ³ H, ¹⁴ C, ⁶⁵ Zn, ⁵⁷ Co and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescein, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Examples of haptens include biotin, digoxigenin, dinitrophenol (DNP), and thymine dimer. These labeling substances can be imparted to the anti-PTX3 antibody by a method known per se. The labeled anti-PTX3 antibody may recognize the same epitope of the anti-PTX3 antibody immobilized on the support and the PTX3 molecule, but preferably recognizes a different epitope.

  After incubation under appropriate conditions, washing is performed again to detect the label remaining on the support. The detection method can be performed by methods known to those skilled in the art. For example, in the case of labeling with a radioisotope, it can be detected by liquid scintillation, RIA method, or the like. In the case of labeling with an enzyme, a substrate is added, and an enzymatic change of the substrate, for example, color development, can be detected with an absorptiometer. As a substrate for peroxidase, for example, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,2-phenylenediamine (ortho-phenylenediamine) and the like, and as a substrate for alkaline phosphatase, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 4-nitroblue tetrazolium chloride (NBT) ) And the like. In the case of a fluorescent substance, it can be detected by a fluorometer.

  Further, as another aspect of the method for examining the presence or amount of PTX3 in the sample-derived component, a detection method utilizing an agglutination reaction can be mentioned. In this method, PTX3 can be detected using a carrier sensitized with an anti-PTX3 antibody. As the carrier for sensitizing the antibody, any carrier may be used as long as it is insoluble, does not cause a non-specific reaction, and is stable. For example, latex particles, bentonite, collodion, kaolin, fixed sheep erythrocytes and the like can be used, but it is preferable to use latex particles. As the latex particles, for example, polystyrene latex particles, styrene-butadiene copolymer latex particles, polyvinyl toluene latex particles and the like can be used, but it is preferable to use polystyrene latex particles. 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 viewing the aggregation with the naked eye. It is also possible to detect turbidity due to aggregation by measuring with a spectrophotometer or the like.

  In the sandwich ELISA method or the detection method utilizing an agglutination reaction, when examining the presence or amount of azulocidin, the antibody against PTX3 can be directly replaced with the antibody against azulocidin, and the ELISA can be carried out in the same manner as described above. When examining the presence or amount of the complex of PTX3 and azulocidin, the anti-PTX3 antibody is immobilized on the support and the labeled anti-azurosidin antibody is used, or vice versa, the anti-azurosidin antibody is immobilized on the support. By using a labeled anti-PTX3 antibody, an ELISA can be performed in the same manner as described above.

  In the detection method of the present invention, the determination of the presence or absence of infection (or the possibility of developing in the future) in step (E) is performed by using the result of step (D) obtained for the test animal as a normal control animal. It can carry out by contrasting with the result of the process (D) obtained about. As a result, when PTX3 and azurosidin are present in the test animal or the amount thereof is increased between the test animal and the control animal, the test animal is affected (or will develop in the future). The possibility is high).

  In the present invention, it can be said that it is important to examine the presence or amount of PTX3 and azulocidin depending on the type of substance used as a test sample (eg, plasma, serum, blood, etc.), for example, By using the detection method of the present invention in combination with PTX3 and azurocidin detected in plasma, PTX3 and azulocidin and the like detected in serum, a more accurate diagnosis of infectious diseases can be performed. In the plasma, as described above, a result corresponding to a test sample treated with heparin, EDTA, or the like can be obtained and combined to be used in a method for detecting the presence or absence of infection. .

3. The present invention also provides a method for simultaneously detecting the presence or absence of an infectious disease and the pathogen of the infectious disease, including the following steps (a) to (g): To do.
(A) contacting a sample collected from a test animal with magnetic particles bound with an anti-pentraxin 3 antibody;
(B) recovering the magnetic particles;
(C) separating the component derived from the sample captured by the anti-pentraxin 3 antibody from the magnetic particles recovered in step (b), and concentrating the component;
(D) a step of performing mass spectrometry of the component concentrated in step (c),
(E) identifying a molecule that binds to pentraxin 3 based on the analysis result of step (d),
(F) Based on the identification result of step (e), a step of examining the presence or amount of pentraxin 3 and azulocidin, and a bacterial or fungal-derived molecule,
(G) A step of detecting presence or absence of an infection and pathogenic bacteria of the infection based on the result of the step (f).

  In the present invention, the steps (a) to (c) correspond to the steps (A) to (C) described above, and the meanings of various terms and the intended contents are the steps (A) to (C). It is the same as that described in.

  Mass spectrometry in the step (d) means that particles such as atoms, molecules, and clusters are converted into gaseous ions by any method (ionization) and moved in a vacuum using electromagnetic force or by time difference of flight. Is used to perform analysis such as separation, detection, and identification of proteins contained in the components concentrated in step (c) according to their mass-to-charge ratio. It is done. In mass spectrometry, a gaseous sample is generally ionized by electron impact or the like, then the ions are separated according to a mass-to-charge ratio (mass / number of charges (m / z)), and the intensity of each ion is recorded to obtain a mass. By obtaining a mass spectrum (mass spectrum) arranged in the order of charge ratios and analyzing the mass spectrum, identification of a known substance, determination of the structure of an unknown substance, and the like can be performed.

  In the present invention, the ionization method is not particularly limited as long as it can analyze the protein contained in the component concentrated in the step (c). For example, an electron impact ionization (EI) method, Chemical ionization (CI) method, Field Ionization (FI) method, Field Desorption (FD) method, Fast Atom Bombardment (FAB) method, Desorption electron ionization (DEI) : Desorption Electron Ionization (MALDI) method, Matrix Assisted Laser Desorption Ionization (MALDI) method, Electrospray Ionization (ESI) method, Atmospheric Pressure Chemical Ionization (APCI) method, frit FRIT-FAB (FRIT-fast atom bom) bardment) method or the like can be used.

  Further, in the present invention, as a method of analyzing these ionized samples, for example, a magnetic field deflection type using ions in a magnetic field and utilizing a change in a flight path due to a Lorentz force received at that time, ions are formed into four electrodes. A quadrupole type (Q: Quadrupole) that perturbs the sample by applying a high-frequency voltage to the electrode and allows only the target ions to pass through, and holds the ions in a trap chamber composed of electrodes. An ion trap type that performs separation by selectively releasing ions by changing the potential, and a time-of-flight type (TOF: Time of Flight) that detects the time difference until the ionized sample is accelerated in a pulse manner and reaches the detector. -of Flight), introducing ions into a cell subjected to an electrostatic field and a static magnetic field, applying a high-frequency voltage to excite the ion motion, and detecting the cycle of the ions, cyclotron Fourier transform ion cyclotron resonance for calculating the mass from the matter (FT-ICR: Fourier-Transform Ion Cyclotron Resonance), (also referred to as MS / MS) These methods tandem that combines the like can be used.

  These mass spectrometry can be performed using an apparatus generally called a mass spectrometer (also referred to as a mass spectrometer). The apparatus has different introduction methods depending on the properties of the sample, that is, whether the sample is a gas or a volatile substance, or a liquid, a solid or a non-volatile substance. For example, high-performance liquid chromatography (HPLC), A mobile phase can be introduced directly connected to gas chromatography (GC), capillary electrophoresis (CE) or the like (referred to as LC / MS, GC / MS, and CE-MS, respectively). About these apparatuses, it does not specifically limit in this invention, What can be purchased commercially can be used. Of the methods related to mass spectrometry, in the present invention, it is possible to analyze a hardly volatile compound or a heat-labile compound, and a plurality of molecules involved in the complex formation of PTX3 and azulocidin and PTX3. Is highly sensitive at a time, and in combination with the above trypsin digestion, it is possible to analyze a small amount of many samples, and therefore, introduction of a test sample by high performance liquid chromatography (LC / MS) is preferable. It is preferable to use electrospray ionization.

  The method for identifying a molecule that binds to PTX3 in step (e) of the present invention is not particularly limited. For example, using a mass spectrometer as described above, the obtained mass spectrum results are registered in advance with known substances. You can do this against the database you have. Examples of such databases include MASCOT search engine (Matrix science), SEQUEST (Thermo Fisher Scientific), X! Examples include Tandem (The Global Proteome Machine Organization) and Phenix (GeneBio). Based on such databases, the presence or absence of PTX3 and azulocidin, and bacterial or fungal-derived molecules can be examined. One feature of the present invention is that by performing mass spectrometry and using such a database, the presence or amount of PTX3 and azulocidin and bacteria or fungi-derived molecules can be examined simultaneously. In addition, as a molecule | numerator couple | bonded with PTX3, the molecule | numerator couple | bonded directly with PTX3 may be sufficient, and it may couple | bond with PTX3 indirectly through another molecule | numerator etc.

  In the present invention, as described above, the step (c ′) may further include a step of decomposing the component concentrated in the step (c) after the step (c). In order to perform mass spectrometry on a component derived from a sample collected from a test animal, particularly a protein component, it is preferable to perform fragmentation with a certain length, and each fragment is also relatively physicochemically uniform. More preferably. For this reason, trypsin digestion is particularly preferable among the methods described above as a method for performing the decomposition in the step (c ′) of the present invention. When trypsin digestion is performed in the present invention, for example, a final concentration of 0.2 to 5.0 ng / min is applied directly to the concentrated component derived from the sample or after the sample derived component is resuspended in a buffer solution or the like. μL trypsin can be added and allowed to react at 30-37 ° C. for 1-16 hours. In addition, when the step (c ′) is used, the step (d) can be a step of performing mass analysis of the component decomposed in the step (c ′).

  In the detection method of the present invention, the presence or absence (or possibility of developing in the future) of the infection in step (g) is determined by obtaining the result of step (f) obtained for the test animal for normal control animals. This can be done by comparing with the result of step (f). As a result, when the PTX3 and azulocidin and bacterial or fungal-derived molecules are present or increased in the test animal between the test animal and the control animal, the test animal suffers from an infectious disease. It is possible to determine the type of bacteria or fungi that can be a pathogen of the infectious disease.

  Further, in the present invention, in the same manner as described above, the presence or amount of PTX3 and azulocidin and bacterial or fungal-derived molecules is determined according to the type of substance (eg, plasma, serum, blood, etc.) used as a test sample. For example, PTX3 and azulocidin detected in plasma, and molecules derived from bacteria or fungi, PTX3 and azulocidin detected in serum, molecules derived from bacteria or fungi, etc. By using the detection method of the invention, it is possible to diagnose infection with higher accuracy. In the plasma, as described above, a result corresponding to a test sample treated with heparin, EDTA or the like is obtained, and a combination thereof is used to simultaneously detect the presence or absence of an infectious disease and a pathogen of the infectious disease. It can also be used.

4). Kit for Diagnosing Infectious Diseases The present invention also provides a kit for diagnosing infectious diseases, comprising a magnetic particle to which an anti-pentraxin 3 antibody is bound, and a substance capable of detecting pentraxin 3 and azulocidin.

  In the present invention, the substance capable of detecting PTX3 and azulocidin is not particularly limited as long as the presence or absence of these two substances or their amounts can be confirmed, but preferably, mass spectrometry of PTX3 and azulocidin Or one or more molecules selected from the group consisting of antibodies, ligands and receptors that recognize PTX3 and azulocidin.

  The internal standard substance that can be used for mass spectrometry of PTX3 and azulocidin is a substance that is used in the so-called internal standard method. For example, when the above-described mass spectrometer is used, the concentration of the substance is measured by this apparatus. A calibration curve can be drawn based on the measured concentration. By using the calibration curve, it is possible to confirm the presence of PTX3 and azulocidin even if the detection output of the apparatus varies depending on the conditions at the time of measurement. Mass can be measured. Examples of these internal standard substances include synthetic peptides specific to amino acid sequences of PTX3 and azulocidin.

  In the present invention, one or more molecules selected from the group consisting of an antibody, a ligand and a receptor that recognize PTX3 and azulocidin can also be adopted as a substance capable of detecting PTX3 and azulocidin. The antibody that recognizes PTX3 and azurocidin may be a single antibody that specifically recognizes both substances simultaneously or a combination of antibodies that specifically recognize both substances. The same applies to ligands and receptors. These anti-PTX3 antibody and anti-azulocidin antibody have the same concept as described above, and can be used, for example, for detection of PTX3 and azulocidin by ELISA. In particular, in the embodiment of the ELISA method, it is preferable to use a labeled anti-PTX3 antibody and a labeled antiazurocidin antibody in combination with these molecules. As the label, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, a hapten, and the like can be used in the same manner as described above, and can be imparted to these antibodies by a method known per se.

  Moreover, when the kit of this invention is based on ELISA method, the support | carrier which solidifies an antibody may be included and the antibody may couple | bond with the support | carrier beforehand. When the kit is based on an agglutination method using a carrier such as latex, the kit may contain a carrier on which an antibody is adsorbed. Further, the kit may appropriately contain a blocking solution, a reaction solution, a reaction stop solution, a reagent for treating the sample, and the like.

[Materials and methods]
1. Preparation of antibody-bound protein G magnetic particles Take 3 mg of protein G magnetic particles (JSR) in a 1.5 mL Eppendorf tube, and twice with antibody reaction buffer (Citrate-Phosphate buffer (pH 5.0), 0.01% Tween 20). Washed. After suspending again in the antibody reaction buffer, 60 μg of anti-PTX3 monoclonal antibody (PPMX0103) was added and reacted at room temperature for 40 minutes with stirring. After removing the supernatant, it was washed twice with an antibody reaction buffer. Further, after washing once with a crosslinking reaction buffer (0.2 M Triethanolamine-HCl (pH 8.2)), a crosslinking reaction buffer containing 20 mM dimethyl pimelimidate was added and reacted at room temperature for 30 minutes with stirring. After removing the supernatant, a reaction stop buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl) was added and allowed to react at room temperature for 15 minutes with stirring. After removing the supernatant, it was washed twice with a washing storage buffer (PBS, 0.01% Tween 20, 0.1% Proline 300), suspended again in the washing storage buffer, and stored at 4 ° C. until use.

2. Immunoseparation of patient plasma and serum PTX3 complex Cryopreserved patient plasma and serum samples were thawed at room temperature, and insoluble components were removed by centrifugation (3000 rpm, 5 minutes) and filtration through a 0.45 μm filter. The sample was passed through a gel filtration column (Superdex ^™ 200 pg, GE Healthcare) equilibrated with PBS, and only the void fraction was collected. NP-40 is added to the collected sample to a final concentration of 0.1%, and 0.5 mg of anti-PTX3 antibody-binding protein G magnetic particles prepared as described above is further added, and the mixture is stirred for 30 minutes. The reaction was performed at room temperature. Subsequently, the anti-PTX3 antibody-binding protein G magnetic particles were collected using a magnet, the supernatant was removed, and then washed 3 times with a washing buffer (PBS, 0.1% NP-40) and once with PBS. Thereafter, an elution buffer (50 mM NH ₄ CO ₃ , 0.05% Rapigest ^™ (WATERS)) was added and incubated at 67 ° C. for 30 minutes. Thereafter, the supernatant was recovered and concentrated by trichloroacetic acid (TCA) / acetone precipitation.

3. Samples concentrated by In-solution digested TCA / acetone precipitation were redissolved in 25% (v / v) CH ₃ CN / 25 mM NH ₄ CO ₃ buffer. Add Tris (2-chloroethyl) phosphate (TCEP) to the final concentration of 0.6 mM in the solution, incubate at 37 ° C for 30 minutes, and then add iodoacetamide to a final concentration of 3 mM. Incubated for 30 minutes at room temperature. Finally, 100 ng trypsin was added, the protein was digested overnight at 37 ° C., dried on Speed Vac (Thermo Fisher), 0.2% TFA / 2% CH ₃ CN was added and at 37 ° C. Redissolved for 1 hour.

4). LC-MS / MS Measurement and Database Search Using the LC-MS / MS system (ZAPLOUS, AMR), the sample obtained as described above was subjected to mass spectrometry of proteins contained in the sample. Data was acquired using data-dependent acquisition mode. The obtained MS / MS data was subjected to database search using MASCOT search engine (Matrix science). Normal database search is limited to trypsin for digested fragments and Homo sapiens (human) for species. When identifying bacterial or fungal proteins, digested fragments are limited to semi-trypsin and species are not limited. Went to.

5). ELISA binding assay Recombinant Azurocidin (R & D Systems) or recombinant C1q (CALBIOCHEM) was prepared at a concentration of 1 μg / mL in TBS, added to a 96-well ELISA plate, and solidified overnight at 4 ° C. The solution in the well was removed, blocking buffer (TBS, 0.1% Triton X-100, 1% BSA) was added and incubated for 2 hours at room temperature. After washing 4 times with a washing buffer (PBS, 0.1% Triton X-100), recombinant PTX3 diluted to various concentrations with a blocking buffer was added to each well and allowed to react at room temperature for 1 hour. After washing 4 times with a washing buffer, an HRP-labeled anti-PTX3 antibody diluted with a blocking buffer was added and reacted at room temperature for 1 hour. After washing 4 times with the washing buffer, a 3,3 ′, 5,5′-tetramethyl-benzidene (TMB) solution was added, a color reaction was performed for 30 minutes, and the absorbance at a wavelength of 450 nm was measured. All buffers were assayed in the presence of 4 mM CaCl ₂ or 4 mM EDTA.

6). BIAcore Measurement In order to examine the intermolecular interaction with PTX3 for the obtained sample, BIAcore was measured using BIAcore 3000 (GE Healthcare). A recombinant PTX3 solution diluted to 100 μg / mL with 10 mM sodium acetate (pH 3.5) was immobilized on a CM5 sensor chip using an amine coupling kit (GE Healthcare). The reaction with recombinant Azurocidin (R & D Systems) was carried out using a predetermined buffer (500 mM NaCl, 20 mM HEPES (pH 7.4), 0.005% surfactant P20) at a flow rate of 20 μL / min. Measurement was performed in the presence of ₂ mM CaCl ₂ or 3 mM EDTA in the buffer. A buffer containing 5 μg / mL of recombinant Azurocidin was passed from 90 seconds to 210 seconds after the start of measurement, and only the buffer was passed from 210 seconds to 330 seconds (FIG. 3). Regeneration of the sensor chip was performed with 10 μL of 1M sodium acetate (pH 7.2) and 10 μL of 10 mM NaOH.

[result]
1. Immunoseparation of PTX3 complex in patient plasma Using EDTA and heparin plasma from septic patients as samples, immunoseparation of PTX3 complex was attempted using anti-PTX3 antibody-bound magnetic particles. As a result of protein staining, the amount of nonspecific protein adsorbed was small, and a specific band considered to be PTX3 and PTX3 binding protein was confirmed (FIG. 1-A). The results of Western blotting confirmed that a sufficient amount of PTX3 was recovered in a specific manner (FIG. 1-B). From the above results, a very small amount of PTX3 complex was recovered from patient plasma with high efficiency. I understood that. This immunoseparation method was judged to be at a level that enables shotgun proteomics analysis.

2. Identification of PTX3 complex in patient plasma and serum Next, an attempt was made to identify a PTX3 complex in patient plasma and serum by shotgun proteomic analysis by LC-MS / MS measurement. For plasma, those treated with heparin and those treated with EDTA were used. The specimens were measured for 4 cases of sepsis and bacteremia. Specifically, case 1 is a patient with pyelonephritis due to E. coli and sepsis, and case 2 is a 57-year-old man who is uncontrollable diabetic, who suffered from sepsis due to foot cresting (negative blood culture) Qualitative procalcitonin 3+), as a case 3, a 94-year-old woman who died of septic shock, with Streptococcus sanguis and Streptcoccus salivarius detected in blood culture, as a case 4, an 82-year-old man with obstructive suppurative The patient had bacteremia due to cholangitis (Pseudomonas aeruginosa and enterococci were detected in blood culture, qualitative procalcitonin 3+). The results of cases 1 to 4 are shown in Tables 1 to 4 below. As a result, Inter-alpha-trypsin inhibitor, Ficolin-2 and Ficolin-3, and Tumor necrosis factor-inducible gene 6 protein were identified. Furthermore, the binding proteins of Mannan-binding lectin serine protease 1 and Mannan-binding lectin serine protease 2 which are Ficolin-2 binding proteins, and the binding proteins of Tumor necrosis factor-inducible gene 6 protein have been identified, Versican core protein and Thrombospondin-1. (Tables 1-4). Various other proteins were identified, and some were identified specifically for the specimen.

3. Identification of PTX3-binding bacterial or fungal derived proteins in patient plasma and serum It was investigated whether bacterial or fungal proteins with potential human infection were identified in PTX3 complexes. As a result, only the plasma treated with heparin was identified for case 1 above, not identified at all for case 2, identified for plasma and serum treated with heparin for case 3, and any test sample for case 4 Among them, bacterial outer membrane proteins (protein F, OprF, hypothetical protein NEILACOT_0259) and Aspergillus fungi (HECT) were found. The above results are shown in Table 5 (Case 1), Table 6 (Case 3), and Table 7 (Case 4), respectively. In addition, the amino acid sequences of the peptides shown in Tables 5 to 7 are shown in SEQ ID NOs: 1 to 22, respectively.

4). Confirmation of binding between PTX3 and Azurocidin Among the identification results of the PTX3 complex protein, Azurocidin was identified only by a specific blood collection method of a specific specimen (Table 1). Since Azurocidin has antibacterial activity and is known to be an inflammatory mediator protein (Wieslaw Watorek Acta Biochimica Polonica 50: 743-752, 2003.), it is expected to function in combination with PTX3. Its binding ability was confirmed. From the results of ELISA binding assay (FIG. 2) and real-time interaction confirmation by BIAcore (FIG. 3), it was confirmed that Azurocidin is a novel protein that binds to PTX3 in a calcium-dependent manner.

  By using the disease marker of the present invention, for example, it is possible to conduct a more accurate examination or diagnosis of an infectious disease simply by collecting blood from a test animal, and also to help early diagnosis of the infectious disease. Can do. Furthermore, if the detection method of the present invention using the disease marker is used, the infectious disease can be efficiently inspected or diagnosed, and the laborious infectious disease inspection or Diagnosis can be made.

Claims (19)

A method for detecting the presence or absence of an infectious disease, comprising the following steps (A) to (E):
(A) a step of contacting a sample collected from a test animal with a carrier bound with an anti-pentraxin 3 antibody;
(B) recovering the carrier;
(C) separating the component derived from the sample captured by the anti-pentraxin 3 antibody from the carrier recovered in step (B), and concentrating the component;
(D) presence or absence of a complex between pentraxin 3 and azurocidin of components to be concentrated in the step (C) or step to examine the amount,
(E) A step of detecting the presence or absence of an infectious disease based on the result of the step (D).   The detection method according to claim 1, wherein the sample collected from the test animal in the step (A) is plasma, serum, or a combination thereof treated with heparin or ethylenediaminetetraacetic acid.   The detection method according to claim 1 or 2, wherein the infectious disease is sepsis.   The detection method according to any one of claims 1 to 3, wherein the carrier in the step (A) is coated with protein G. The detection method according to any one of claims 1 to 4, further comprising a step (C ') after the step (C):
(C ′) A step of decomposing the component concentrated in step (C).   The detection method according to claim 5, wherein the degradation in step (C ') is trypsin digestion.   The presence or absence of pentraxin 3 and azulocidin is obtained by comparing the result of the step (D) obtained for the test animal with respect to the result of the step (D) obtained by the determination of the presence or absence of the infectious disease in the step (E). The detection method according to any one of claims 1 to 6, wherein the detection method is carried out using as an indicator that the amount is increased or the amount thereof is increasing. Simultaneous detection method of presence or absence of infection and pathogenic bacteria of infection including the following steps (a) to (g):
(A) a step of contacting a sample collected from a test animal with a carrier bound with an anti-pentraxin 3 antibody;
(B) recovering the carrier;
(C) separating the component derived from the sample captured by the anti-pentraxin 3 antibody from the carrier recovered in step (b) and concentrating the component;
(D) a step of performing mass spectrometry of the component concentrated in step (c),
(E) identifying a molecule that binds to pentraxin 3 based on the analysis result of step (d),
(F) based on the identification result of step (e), a step of examining the presence or absence or amount of a complex of pentraxin 3 and azulocidin, and a bacterial or fungal-derived molecule;
(G) A step of detecting presence or absence of an infection and pathogenic bacteria of the infection based on the result of the step (f).   The detection method according to claim 8, wherein the sample collected from the test animal in the step (a) is plasma, serum or a combination thereof treated with heparin or ethylenediaminetetraacetic acid.   The detection method according to claim 8 or 9, wherein the infectious disease is sepsis.   The detection method according to any one of claims 8 to 10, wherein the carrier in the step (a) is coated with protein G. The detection method according to any one of claims 8 to 11, further comprising a step (c ') after the step (c).
(C ′) A step of decomposing the component concentrated in step (c).   The detection method according to claim 12, wherein the degradation in step (c ') is trypsin digestion.   The determination of the presence or absence of infection in step (g) and the pathogen of the infection is obtained by comparing the result of step (f) obtained for the test animal with the result of step (f) obtained for the normal control. The detection method according to any one of claims 8 to 13, which is performed using 3 and azulocidin as well as the presence of a bacterium or fungal-derived molecule or an increase in the amount thereof. An infectious disease diagnosis kit comprising a carrier bound with an anti-pentraxin 3 antibody, and a substance capable of detecting a complex of pentraxin 3 and azulocidin.   The diagnostic kit according to claim 15, wherein the carrier is coated with protein G. Substance capable of detecting a complex between pentraxin 3 and azurocidin are internal standard that may be used in the mass spectrometry of pentraxin 3 and azurocidin, or pentraxin 3 and azurocidin recognizing antibody is selected from the group consisting of ligands and receptors The diagnostic kit according to claim 15 or 16, which is one or more molecules. Pentraxin 3 and Ri name contains azurocidin, and pentraxin 3 and azurocidin are obtained by forming a complex disease marker of infection. The disease marker according to claim 18 , wherein the infectious disease is sepsis.