WO2012108538A1 - 急性肺損傷診断方法 - Google Patents
急性肺損傷診断方法 Download PDFInfo
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- WO2012108538A1 WO2012108538A1 PCT/JP2012/053174 JP2012053174W WO2012108538A1 WO 2012108538 A1 WO2012108538 A1 WO 2012108538A1 JP 2012053174 W JP2012053174 W JP 2012053174W WO 2012108538 A1 WO2012108538 A1 WO 2012108538A1
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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
- G01N33/6884—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from lung
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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
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to a test method for diagnosing acute lung injury, a diagnostic agent for acute lung injury, and the like.
- Acute lung injury also called Acute Lung Injury; ALI
- ALI Acute Lung Injury
- ALI Acute Lung Injury
- patients with acute lung injury typically begin with the above symptoms within 24-48 hours of the original injury or disease, and in severe cases death. Mortality is very high, 30-40%, and is further increased by aging or comorbidities.
- Diagnosis of acute lung injury is basically made by clinical diagnosis. Because clinical findings of acute lung injury are similar to those of acute heart failure and pulmonary infection, in order to confirm the diagnosis and identify the cause, the patient is further asked to use ABG (Arterial Blood Gas; Blood oxygen pressure, CO 2 partial pressure, pH, etc. are measured) and chest X-ray examination should be performed.
- ABG Arterial Blood Gas; Blood oxygen pressure, CO 2 partial pressure, pH, etc. are measured
- acute lung injury is a disease with a very poor prognosis that progresses rapidly and no effective treatment has been established. Complications such as pneumothorax and severe bacterial infections of the lung may occur. Therefore, particularly early diagnosis is required.
- serum markers such as KL-6 (sialylated glycan antigen), SP-D (Surfactant protein-D), and SP-A (Surfactant protein-A) have been used to more easily and quickly diagnose acute lung injury. It has come to be utilized.
- idiopathic organizing pneumonia cryptogenic organizing pneumonia; oniaCOP
- idiopathic normal interstitial pneumonia idiopathic usual interstitial pneumonia; idiopathic UIP
- idiopathic nonspecific interstitial pneumonia idiopathic nonspecific interstitial pneumonia; idiopathic (NSIP)
- Collagen-vascular interstitial pneumonia collagen-vascular-disease- (CVD) -associated UIP
- non-specific interstitial pneumonia associated with collagen disease collagen-vascular-disease- (CVD) -associated-NSIP
- Heat shock protein 47 is a molecular chaperone having specificity for collagen, and is known as a heat shock-inducing stress protein present in the endoplasmic reticulum. Since the expression of HSP47 is coupled with the expression of collagen as a substrate, it has been pointed out that HSP47 has an important relationship in various fibrotic diseases (Patent Documents 1 to 3, Non-Patent Documents 6 and 8). . The role of HSP47 in the fibrosis phase of acute lung injury has also been reported (Non-Patent Documents 1 to 3).
- Non-Patent Documents 5 and 6 HSP47 expression is increased in the lung tissue of pulmonary fibrosis (Non-Patent Documents 5 and 6), but HSP47 is localized in the endoplasmic reticulum and does not leak out of the cell. Therefore, in interstitial pneumonia with fibrosis, it was not considered that HSP47 was elevated extracellularly (for example, in the blood, etc.). In fact, past reports have revealed that HSP47 in the serum of patients with idiopathic pulmonary fibrosis is at the same level as that of healthy individuals and no increase is observed (Non-patent Document 7). It has never been known that HSP47 is a useful biomarker with a marked increase in acute lung injury and a clear distinction between acute lung injury and chronic progressive interstitial pneumonia. It was not and was not expected.
- An object of the present invention is to provide a useful test method and diagnostic agent capable of clearly distinguishing and diagnosing acute lung injury from chronic progressive interstitial pneumonia.
- the present inventors have intensively studied to solve the above problems, and that the measurement (concentration) of HSP47 protein in a biological sample is useful for diagnosis of acute lung injury, and that HSP47 protein in a biological sample is chronically progressive. As compared with interstitial pneumonia, a specific increase was observed in acute lung injury, and it was found useful to distinguish these, and the present invention was completed.
- the present invention is as follows. [1] A method for determining whether or not a subject suffers from acute lung injury, comprising quantifying heat shock protein 47 in a biological sample derived from the subject. [2] The method according to [1], which is a method for distinguishing between acute lung injury and chronic progressive interstitial pneumonia.
- a method for determining whether or not a subject develops acute lung injury comprising quantifying heat shock protein 47 in a biological sample derived from the subject.
- the inspection method of the present invention it is possible to determine with high sensitivity whether or not a subject suffers from acute lung injury.
- the method can be a powerful diagnostic tool that can clearly differentiate between chronic progressive interstitial pneumonia and acute lung injury. Furthermore, if the diagnostic agent of this invention is used, it can be easily diagnosed whether it suffers from acute lung injury with the test
- FIG. 1 is a graph showing the HSP47 protein concentration in the serum of healthy subjects and patients with each disease described on the horizontal axis.
- FIG. 2 is a graph showing the KL-6 concentration in the serum of healthy subjects and patients with each disease described on the horizontal axis.
- FIG. 3 is a graph showing the SP-A concentration in the serum of healthy subjects and patients with each disease described on the horizontal axis.
- FIG. 4 is a graph showing the SP-D concentration in the serum of healthy subjects and patients with each disease described on the horizontal axis.
- FIG. 5 is a graph showing LDH (lactate dehydrogenase) concentrations in the serum of healthy subjects and patients with each disease described on the horizontal axis.
- FIG. 1 is a graph showing the HSP47 protein concentration in the serum of healthy subjects and patients with each disease described on the horizontal axis.
- FIG. 2 is a graph showing the KL-6 concentration in the serum of healthy subjects and patients with each disease described on the horizontal axis.
- FIG. 3 is
- FIG. 6 is a graph showing the relationship between sensitivity and specificity for the HSP47 protein, KL-6, SP-D, SP-A, and LDH by a receiver operating characteristic curve (Receiver Operating Characteristic (ROC) curve).
- FIG. 7 is a graph showing the HSP47 protein concentration in the serum of healthy subjects and patients with each disease described on the horizontal axis.
- FIG. 8 is a graph showing the KL-6 concentration in the serum of healthy subjects and patients with each disease described on the horizontal axis.
- FIG. 9 is a graph showing the SP-A concentration in the serum of healthy subjects and patients with each disease described on the horizontal axis.
- FIG. 10 is a graph showing the SP-D concentration in the serum of healthy subjects and patients with each disease described on the horizontal axis. N.S.
- FIG. 11 is a graph showing LDH (lactate dehydrogenase) concentrations in the serum of healthy subjects and patients with each disease described on the horizontal axis.
- FIG. 12 is a graph showing the relationship between sensitivity and specificity for the HSP47 protein, KL-6, SP-D, SP-A, and LDH by a receiver operating characteristic curve.
- heat shock protein 47 as a biomarker for diagnosis of acute lung injury
- the present invention comprises quantifying heat shock protein 47 in a biological sample derived from a subject, wherein the subject suffers from acute lung injury.
- a method (method (1)) for determining whether or not there is a problem is provided.
- acute lung injury refers to an inflammatory disease of the lung caused by sepsis, pneumonia, trauma, and / or aspiration that directly or indirectly injures the lung.
- Acute lung injury herein includes acute respiratory distress syndrome (also referred to as ARDS), which is known to be the same clinical disorder. Rapid lung interstitial pneumonia that satisfies the clinical definition of acute lung injury is also included in acute lung injury.
- the “subject” may be a “subject suspected of suffering from acute lung injury”.
- “subject suspected of suffering from acute lung injury” refers to acute lung injury, aortic dissection, acute heart failure, acute exacerbation of chronic heart failure, diffuse alveolar hemorrhage, cancer Clinical findings (eg, cough, sputum, fever, dyspnea, breathing) common to systemic lymphangiopathy, re-expansion pulmonary edema, pulmonary edema due to excessive infusion, neurogenic pulmonary edema, or pulmonary infection (pulmonary tuberculosis, miliary tuberculosis, etc.) Refers to patients who have acquired insufficiency, hypoxemia, chest pain, etc. (these diseases other than acute lung injury may be collectively referred to as “the above-mentioned diseases with common clinical findings with acute lung injury”). is there).
- the “subject” or “subject suspected of suffering from acute lung injury” suffers from either “acute lung injury or chronic progressive interstitial pneumonia”.
- a subject suspected of having In the present specification, “subject suspected of suffering from either acute lung injury or chronic progressive interstitial pneumonia” refers to a chest radiograph, KL-6, SP-D or SP- This refers to patients who have been diagnosed with a serum marker such as A and suspected of suffering from either acute lung injury or chronic progressive interstitial pneumonia.
- interstitial pneumonia refers to a disease causing inflammation mainly in the stromal tissue of the lung, which is characterized by alveolar septum thickening, fibroblast proliferation, collagen deposition, and the like.
- Examples of interstitial pneumonia include those caused by radiation, pathogen infection, and collagen disease, poisoning / drug-related ones, and idiopathic ones where no clear cause can be found.
- interstitial pneumonia examples include idiopathic organizing pneumonia (cryptogenic organizing pneumonia; COP), idiopathic normal interstitial pneumonia (idiopathic usual interstitial pneumonia; idiopathic UIP), idiopathic nonspecific interstitial Pneumonia (idiopathic idnonspecific interstitial pneumonia; idiopathic NSIP) and the like are included, but not limited thereto.
- idiopathic organizing pneumonia cryptogenic organizing pneumonia
- COP idiopathic normal interstitial pneumonia
- idiopathic UIP idiopathic nonspecific interstitial Pneumonia
- interstitial pneumonia is a concept including pulmonary fibrosis in which an inflamed tissue has progressed to fibrosis.
- the subject subjected to the method (1) of the present invention may or may not suffer from pulmonary fibrosis. So far, it has been reported that HSP47 expression is increased in the lung tissue of pulmonary fibrosis (Non-Patent Documents 5 and 6), but HSP47 is localized in the endoplasmic reticulum and does not leak out of the cell. Therefore, in interstitial pneumonia with fibrosis, it was not considered that HSP47 was elevated extracellularly (for example, in the blood, etc.). In fact, past reports have revealed that HSP47 and anti-HSP47 antibodies in the serum of patients with idiopathic pulmonary fibrosis are at the same level as that of healthy individuals and no increase is observed (Non-patent Document 7). Extracellular (eg, in the blood) HSP47 is not elevated in chronic progressive interstitial pneumonia but is specifically elevated in acute lung injury (including rapidly progressive interstitial pneumonia).
- the “subject” or “subject suspected of suffering from acute lung injury” is “a clinical finding common to acute lung injury or acute lung injury” A subject suspected of suffering from any of the above-mentioned diseases.
- “subject suspected of suffering from any of the above-mentioned diseases that have common clinical findings with acute lung injury or acute lung injury” refers to acute lung injury and the above-mentioned diseases by clinical diagnosis. Refers to patients who have common clinical findings (eg, dyspnea, respiratory failure, hypoxemia, chest pain, etc.).
- aortic dissection means that a part of the intima of the aortic blood vessel is torn due to genetic factors, aortic intima degeneration and weakening, as well as aortic dilatation and hypertension. It refers to a disease accompanied by sudden chest pain and abdominal pain that develops when blood flows from the intimal hiatus into the intima causing dissection of the blood vessel wall.
- acute heart failure refers to a pathological condition of a circulatory disease in which sufficient blood flow is not supplied from the ventricle to a peripheral organ, and the pathological condition is deteriorated rapidly, resulting in the pathological condition.
- Symptoms of acute heart failure include significant dyspnea based on pulmonary congestion or cardiogenic shock based on low cardiac output, foaming, oliguria / anuria, cold limbs, decreased blood pressure, cold sweat or tachycardia ( (Sometimes bradycardia) and the like.
- chronic heart failure acute exacerbation means that chronic heart failure rapidly deteriorates.
- chronic heart failure means an equilibrium state in which various compensatory mechanisms such as cardiac hypertrophy are working gradually as seen in old myocardial infarction, dilated cardiomyopathy, etc. This refers to a condition that causes symptoms such as shortness of breath, feeling of fatigue, decreased exercise tolerance, hepatic spleen enlargement, edema, or peripheral vein anger.
- pulmonary tuberculosis refers to pulmonary infection caused by Mycobacterium tuberculosis.
- microtubule tuberculosis refers to a pathological condition in which a large amount of tuberculosis bacteria enter the bloodstream and disseminated in multiple organs to form a large number of tuberculosis nodules.
- “diffuse alveolar hemorrhage” refers to bleeding into the alveolar space. Bleeding into the alveolar space, appearance of hemosiderin phagocytic cells (iron-bearing cells) in the alveolar space, hemosiderin deposition on the alveolar wall, chest radiograph showing diffuse dispersive shadow over the entire lung field Alveolar hemorrhage syndrome is known as a disease group to be shown. Many have bleeding not only in the alveolar space but also in the lung interstitium. Lung damage and chest compression, vascular breakdown of lung-bronchial lesions, vascular nerve bleeding, some hemorrhagic pneumonia, leaky bleeding in blood diseases, bleeding from capillary disorders, bleeding related to immune phenomenon, etc. Can be cited as the cause.
- cancer lymphangiopathy refers to cancer cells that have proliferated in a retrograde manner due to lymph flow stasis due to cancer cell metastasis to lymph nodes on the perfusion side or lymphatic embolization.
- lymphatic embolization Refers to the state of filling and spreading in the lymphatic vessels of the tissue. Enlarged lymphatic peripheries are accompanied by edema and fibrous tissue growth, showing a pseudo-inflammatory condition reminiscent of lymphangitis. It may be found in breast cancer, stomach cancer, and lung cancer including metastasis, and has a poor prognosis.
- pulmonary edema refers to a pathological condition in which abnormal water retention occurs outside the pulmonary blood vessels.
- re-expanding pulmonary edema means that when the pleural effusion, pneumothorax, hemothorax, etc. are treated with thoracic drainage, the lungs that had collapsed suddenly re-expanded. Pulmonary edema that is thought to occur as a result of reperfusion and increased vascular permeability.
- infusion refers to a treatment method in which water or electrolytes are administered by intravenous infusion
- pulmonary edema due to excessive infusion refers to pulmonary capillaries due to excessive infusion. A condition in which pressure increases and pulmonary edema is caused.
- neurogenic pulmonary edema refers to acute pulmonary edema that occurs with increased intracranial pressure, such as head trauma, epileptic seizures, and cerebrovascular disorders.
- the subject subjected to the method (1) of the present invention means a mammal.
- the mammal is not particularly limited as long as it is a mammal that may suffer from acute lung injury.
- laboratory animals such as rodents such as mice, rats, hamsters, guinea pigs, and rabbits, pigs, cows, etc.
- domestic animals such as goats, horses, sheep and minks, pets such as dogs and cats, primates such as humans, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees are preferred, and humans are particularly preferred.
- Heat shock protein 47 is a known heat shock protein, and its amino acid sequence is also known.
- HSP47 used in the present invention is usually derived from a mammal.
- “Mammalian origin” means that the amino acid sequence of HSP47 is a mammalian sequence. Examples of mammals include the same mammals that can be used as subjects.
- the type of mammal from which HSP47 to be quantified is derived is usually the same as the type of mammal of the subject. For example, if the subject is a human, human heat shock protein 47 is quantified.
- amino acid sequence represented by SEQ ID NO: 2 (GeneBank accession number: NP_001226) can be exemplified.
- proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation.
- biological samples examples include blood, bronchial lavage fluid, alveolar lavage fluid, sputum, lung tissue, cerebrospinal fluid, ascites, and the like. There is no particular limitation as long as an increase can be detected.
- the lung tissue is preferably subjected to the method (1) of the present invention by crushing it in an appropriate buffer solution or the like in the form of an extract.
- blood blood derived from any tissue can be assumed, but peripheral blood is usually used because of easy collection.
- a blood collection method a method known per se can be applied.
- the collected blood may be used in this step as it is, but it is used as a liquid component (plasma) from which cell components (red blood cells, white blood cells, platelets, etc.) have been separated using methods known per se, such as centrifugation and filtration. It is preferable to use in the process. It is also preferable to use in this step as a liquid component (serum) obtained by coagulating blood to separate platelets and coagulation factors.
- the biological sample that can be used in the method (1) of the present invention is preferably blood, serum or plasma.
- Quantification of HSP47 in a biological sample can be performed by a method known per se such as an immunological technique or mass spectrometry.
- Quantification of HSP47 by immunological techniques includes, for example, the following steps: (1) A step of bringing a biological sample derived from a subject into contact with an antibody that specifically recognizes HSP47 to form a complex of HSP47 in the biological sample and an antibody that specifically recognizes HSP47; (2) a step of detecting the complex formed in the step (1); and (3) a step of calculating the amount of HSP47 in the biological sample from the amount of the complex detected in the step (2).
- Step (1) is a step in which a biological sample derived from a subject is brought into contact with an antibody that specifically recognizes HSP47 to form a complex of HSP47 in the biological sample and an antibody that specifically recognizes HSP47. is there.
- the “antibody specifically recognizing HSP47” only needs to have the ability to specifically bind to HSP47, and may be either a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable.
- Such antibodies also include binding fragments such as chimeric antibodies, single chain antibodies, or F (ab ′) 2 , Fab ′, or Fab fractions of antibody molecules.
- antibodies prepared by a method known per se using HSP47 as an immunogen can be used, and commercially available antibodies can also be used.
- Specific means that the affinity of the antibody for HSP47, which is an antigen, is higher than the affinity for other antigens.
- recombinant HSP47 When recombinant HSP47 is used as an antigen, recombinant HSP47 can be produced, for example, by the following method.
- a polynucleotide encoding the amino acid sequence of HSP47 (for example, in the case of human HSP47, a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 (GeneBank accession number: NN_001235)) is incorporated into an appropriate expression vector,
- the desired recombinant HSP47 can be obtained from a transformed cell by inserting it into an appropriate host and transforming it.
- the host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, Bacillus subtilis, yeast, plant or animal cells can be used.
- HSP47 may be isolated or purified from a natural cell that produces the protein by a known protein separation and purification technique. Alternatively, it may be a protein synthesized chemically or biochemically by a cell-free translation system.
- the above-mentioned “antibody specifically recognizing HSP47” can be produced according to a conventional method. Specifically, when the antibody is a polyclonal antibody, HSP47 can be immunized to rabbits and other non-human animals according to a conventional method, and can be obtained from the sera of the immunized animal (Current Protocols in Molecular Biology, edit. Ausubel FM et al. (2011) Publish. John Wiley and Sons. Chapter 11, Section III, Unit 11.12 ⁇ 11.13).
- the antibody specifically recognizing HSP47 used in the present invention is preferably isolated or purified. “Isolated or purified” means that an operation for removing components other than the target component from a natural state has been performed.
- the purity of the antibody that specifically recognizes isolated or purified HSP47 (the ratio of the weight of the antibody that specifically recognizes HSP47 to the total protein weight) is usually 50% or more, preferably 70% or more, more preferably Is 90% or more, most preferably 95% or more (eg, substantially 100%).
- the antibody may be directly or indirectly labeled with a labeling substance.
- a labeling substance e.g., FITC, rhodamine
- a radioactive material e.g., 14 C, 3 H, 125 I
- enzymes e.g., alkaline phosphatase, peroxidase
- colored particles e.g., metal colloid particles, colored Latex
- biotin e.g., biotin and the like.
- only one type of antibody may be used in this step, or two or more types may be used.
- the above-mentioned “antibody specifically recognizing HSP47” can be used in the form of an aqueous solution, but is preferably bound to a solid phase.
- solid phases include plates (eg, microwell plates), tubes, beads (eg, plastic beads, magnetic beads), chromatographic carriers (eg, Sepharose TM), membranes (eg, nitrocellulose membrane) , PVDF film), gel (eg, polyacrylamide gel), metal film (eg, gold film) and the like.
- plates, beads, membranes, and metal films are preferably used, and plates are most preferably used because of easy handling.
- the bond include covalent bond, ionic bond, physical adsorption, and the like, and are not particularly limited.
- the solid phase may be directly bonded to the solid phase or indirectly bonded to the solid phase using a substance known per se. Furthermore, in order to suppress non-specific adsorption and non-specific reaction, a phosphate buffer solution such as bovine serum albumin (BSA) or bovine milk protein is brought into contact with the solid phase, and the solid surface portion not coated with the antibody is removed. Blocking with the BSA or bovine milk protein is generally performed.
- BSA bovine serum albumin
- bovine milk protein Blocking with the BSA or bovine milk protein is generally performed.
- the contact between the “antibody specifically recognizing HSP47” in this step and “HSP47” contained in the subject-derived biological sample specifically recognizes the biological sample and HSP47 in the reaction container.
- the embodiment, order, specific method and the like are not particularly limited.
- the contact is made, for example, by adding the biological sample (extract thereof) to a plate on which “an antibody that specifically recognizes HSP47” is immobilized.
- the time for maintaining such contact is particularly sufficient if the antibody that specifically recognizes the HSP47 and the HSP47 contained in the biological sample derived from the subject bind to form a complex. Although it is not limited, it is usually several seconds to several tens of hours, and preferably 1 minute to 2 hours, most preferably 2 minutes to 30 minutes, from the viewpoint of promptly determining whether or not acute lung injury has occurred. .
- the temperature condition for the contact is usually 4 ° C. to 50 ° C., preferably 4 ° C. to 37 ° C., and most preferably room temperature of about 15 ° C. to 30 ° C.
- the pH condition for carrying out the reaction is preferably 5.0 to 9.0, particularly preferably in the neutral range of 6.0 to 8.0.
- Step (2) is a step of determining whether HSP47 is present in the biological sample by detecting the complex formed in the step (1).
- the above detection is carried out by detecting “HSP47” or “an antibody that specifically recognizes HSP47” contained in the complex.
- This detection includes enzyme immunoassay (EIA), immunochromatography, latex agglutination, radioimmunoassay (RIA), fluorescence immunoassay (FIA), luminescence immunoassay, surface plasmon resonance ( SPR method) can be used.
- EIA enzyme immunoassay
- RIA radioimmunoassay
- FIA fluorescence immunoassay
- SPR method surface plasmon resonance
- the EIA method, immunochromatography method, FIA method and SPR method are preferable from the viewpoint of ease of operation and rapidity.
- the EIA method is a sandwich enzyme-linked immunosorbent assay (sandwich ELISA method) using two types of “antibodies that specifically recognize HSP47”. Preferably there is.
- sandwich ELISA method is excellent in specificity to an antigen because it uses two types of antibodies.
- the two types of “antibodies that specifically recognize HSP47” for sandwich ELISA are combinations of monoclonal antibodies, combinations of polyclonal antibodies, or monoclonal antibodies as long as they are combinations of antibodies that do not compete for epitopes. And any combination of polyclonal antibodies.
- a method using avidin-biotin reaction is applicable as a kind of sandwich ELISA method.
- HSP47 in plasma or serum is captured with any solid-phased “antibody that specifically recognizes HSP47”, and the captured HSP47 and biotin-labeled “HSP47 are specifically recognized.
- An antigen-antibody reaction is performed between the antibody and the antibody.
- the time required for such a reaction is preferably 1 minute to 2 hours, more preferably 2 minutes to 30 minutes, from the viewpoint that rapid measurement is required.
- enzyme-labeled streptavidin is added to cause the avidin-biotin reaction.
- the time required for such a reaction is preferably 5 minutes to 1 hour, more preferably 15 minutes to 30 minutes, from the viewpoint that rapid measurement is required.
- biotin label “an antibody that specifically recognizes HSP47” can be produced by binding biotin and “an antibody that specifically recognizes HSP47” by a method known per se.
- biotin and “an antibody that specifically recognizes HSP47” can be bound using a commercially available biotin labeling kit.
- a commercially available enzyme-labeled streptavidin can be preferably used.
- a sandwich ELISA method using an enzyme-labeled antibody is also applicable.
- HSP47 in blood is captured by any solid-phased "antibody specifically recognizing HSP47", and the captured HSP47 and enzyme-labeled "antibody specifically recognizing HSP47"
- An antigen-antibody reaction is performed between The time required for such a reaction is preferably 1 minute to 2 hours, more preferably 2 minutes to 30 minutes, from the viewpoint that rapid measurement is required.
- HSP47 is detected by detecting this enzyme.
- the enzyme-labeled antibody can be produced by binding (labeling) an enzyme and “an antibody that specifically recognizes HSP47” by a method known per se, such as the glutaraldehyde method or the maleimide method.
- a sandwich ELISA method using a secondary antibody is also applicable from the viewpoint of versatility.
- HSP47 in blood is captured by any solid-phased “antibody specifically recognizing HSP47”, and the captured HSP47 is derived from a different animal species from the solid-phased antibody.
- An antigen-antibody reaction is carried out with “an antibody that specifically recognizes HSP47” (referred to as the primary antibody in this paragraph).
- the time required for such a reaction is preferably 1 minute to 2 hours, more preferably 2 minutes to 30 minutes, from the viewpoint that rapid measurement is required.
- the primary antibody is reacted with an antibody derived from an animal species other than a rabbit, for example, a mouse.
- an antigen-antibody reaction is performed between the primary antibody and the enzyme-labeled “antibody recognizing the primary antibody” (described as a secondary antibody in this paragraph).
- the time required for such a reaction is preferably 1 minute to 2 hours, more preferably 2 minutes to 30 minutes, from the viewpoint that rapid measurement is required.
- HSP47 is detected by detecting this enzyme.
- a commercially available enzyme-labeled secondary antibody can be preferably used.
- enzyme-labeled streptavidin and enzyme-labeled antibody examples include peroxidase, alkaline phosphatase, glucose oxidase, ⁇ -galactosidase and the like.
- an appropriate one is selected according to the selected labeling enzyme. For example, when peroxidase is selected as the enzyme, o-phenylenediamine (OPD), tetramethylbenzidine (TMB) or the like is used. When alkaline phosphatase is selected, p-nitrophenyl phosphate (PNPP) or the like is used. used. As the reaction stop solution and substrate solution, conventionally known ones can be appropriately used without particular limitation depending on the selected enzyme.
- step (1) HSP47 in blood derived from a subject is bound to a solid phase in the same manner as described above, and then an antigen-antibody reaction is performed with the labeled “antibody specifically recognizing HSP47”. To form a complex.
- the time required for such a reaction is preferably 1 minute to 2 hours, more preferably 2 minutes to 30 minutes, from the viewpoint that rapid measurement is required.
- HSP47 can be detected using a technique according to the label.
- the “antibody specifically recognizing HSP47” immobilized in a line on a water-absorbing substrate such as a nitrocellulose membrane For example, HSP47 is captured by developing blood, and an antigen-antibody reaction is performed between the captured HSP47 and the labeled “antibody specifically recognizing HSP47”.
- the time required for such a reaction is preferably 1 minute to 2 hours, more preferably 2 minutes to 30 minutes, from the viewpoint that rapid measurement is required.
- HSP47 can be detected using a technique according to the label.
- antibodies that specifically recognize HSP47 are also combinations of monoclonal antibodies, combinations of polyclonal antibodies, or monoclonal antibodies as long as they are combinations of antibodies that do not compete for epitopes. Any combination of polyclonal antibodies may be used.
- the FIA method is selected as the detection method in step (2), the same method as described above is used, using an antibody in which the enzyme bound to the “antibody specifically recognizing HSP47” used in the EIA method is replaced with a fluorescent substance. Perform a sandwich ELISA. Subsequently, HSP47 is detected by detecting a fluorescent substance using a commercially available measuring instrument, a fluorescence microscope, a confocal microscope, etc.
- the fluorescent substance chemical substances such as APC, PE, Cy2, Cy3, Cy5, ECD, FITC, PerCP, Alexa (registered trademark) Fluor, fluorescein, and rhodamine can be preferably used.
- the chemical substance can be labeled on the antibody by a method known per se.
- the antibody on the surface of the metal film (sensor chip) on which the antibody specifically recognizing HSP47 is solid-phased and the blood flowing on the surface of the metal film Interaction with HSP47 is detected as a time course of surface plasmon resonance.
- the “antibody specifically recognizing HSP47” to be immobilized on the metal film to be the sensor chip may be one type, which may be a monoclonal antibody or a polyclonal antibody. Good.
- a measuring instrument used for the detection of SPR a commercially available measuring instrument can be preferably used.
- the EIA method when the EIA method is applied, blood collected from a subject is shaken at room temperature for a certain period of time, and then centrifuged to obtain serum. Next, this serum is dispensed onto a microplate on which any “antibody specifically recognizing HSP47” is immobilized, and left at room temperature for a certain period of time. The plate is washed to remove unreacted antigen, and then the biotinylated antibody solution is dispensed onto the plate and allowed to stand for a certain time to form a complex. Further, the plate is washed to remove unreacted antibodies, and then a peroxidase-labeled streptavidin solution is dispensed onto the plate and reacted at room temperature for a predetermined time. After washing the plate, the complex is detected by reacting with a chromogenic substrate solution such as TMB.
- a chromogenic substrate solution such as TMB.
- the serum or serum diluted with physiological saline or the like is immersed in a test piece and developed.
- the test piece is formed by laminating a granular label holding carrier and a carrier for absorbing a liquid sample made of filter paper on one end of a strip-shaped antibody-immobilized support through one end.
- a water-absorbing carrier made of filter paper is laminated on one upper end side through one end.
- the above-mentioned antibody-immobilized support is one in which an “antibody specifically recognizing HSP47” that undergoes an antigen-antibody reaction with HSP47 is immobilized on a nitrocellulose sheet.
- the solid phase is formed by applying the antibody solution onto a nitrocellulose sheet and drying.
- the immobilized antibody specifically recognizes HSP47 in the developed serum, and can capture a complex of HSP47 and the above-mentioned antibody that is particulate-labeled. Therefore, if “an antibody that specifically recognizes HSP47” is immobilized on a line on an antibody-immobilized support, the line will be colored with a granular label, and HSP47 is present in the blood. Can be judged.
- step (3) the amount of HSP47 in the biological sample is calculated from the amount of the complex detected in step (2).
- the amount (concentration) of the complex detected in the step (2) can be measured by applying a signal such as fluorescence intensity or absorbance to a separately prepared calibration curve.
- concentration of HSP47 in the subject's biological sample can be quantified by multiplying the concentration obtained from the calibration curve by the dilution factor.
- the calibration curve has the sample concentration obtained by serial dilution of the standard HSP47 protein preparation as the horizontal axis (vertical axis), and the signal of the antibody that specifically recognizes HSP47 bound to HSP47 in the standard HSP47 protein preparation in arbitrary units.
- the measured values are plotted on the vertical axis (horizontal axis) indicated by, and are represented by approximate equations.
- the calibration curve can be approximately expressed as a straight line by logarithmically displaying both axes.
- the amount of HSP47 in a biological sample from the subject is higher in patients with acute lung injury than in healthy subjects and patients with chronic progressive interstitial pneumonia.
- the amount of HSP47 in the biological sample is higher in patients with acute lung injury than in patients with the above-mentioned diseases that have common clinical findings with acute lung injury. The above determination is made based on a positive correlation between the amount of HSP47 in a biological sample derived from such a subject and the likelihood of suffering from acute lung injury.
- a biological sample eg, serum
- a biological sample eg, serum
- negative control chronic progressive interstitial pneumonia
- positive control positive control
- HSP47 in the biological sample collected from the subject Is compared to that of the positive and negative controls.
- a correlation diagram between the amount of HSP47 in a specific biological sample (eg, serum) and the prevalence of acute lung injury is prepared in advance, and the amount of HSP47 in a biological sample collected from a subject is compared with the correlation diagram. May be.
- the comparison of the amount of HSP47 is preferably performed based on the presence or absence of a significant difference.
- a cut-off value of the amount of HSP47 in the biological sample is set in advance, and the measured amount of HSP47 can be compared with this cut-off value. For example, when the amount of HSP47 in a biological sample derived from a subject is equal to or higher than the cut-off value, it can be determined that the subject is highly likely to suffer from acute lung injury. Conversely, if the amount of HSP47 in the biological sample derived from the subject is below the cut-off value, it can be determined that the subject is unlikely to suffer from acute lung injury.
- the amount of HSP47 in a biological sample derived from a subject is equal to or higher than the cut-off value. Can be determined that the subject is not chronically progressive interstitial pneumonia and is relatively likely to have acute lung injury. Conversely, if the amount of HSP47 in the subject-derived biological sample is below the cut-off value, the subject is likely not suffering from acute lung injury but suffering from chronic progressive interstitial pneumonia Can be determined.
- the “cut-off value” is a value that can satisfy both high diagnostic sensitivity (prevalence of prevalence of disease) and high specificity of diagnosis (prevalence of nondiagnosticity) when determining the disease or condition based on that value. is there.
- high diagnostic sensitivity prevalence of prevalence of disease
- high specificity of diagnosis prevalence of nondiagnosticity
- the amount of HSP47 that shows a high positive rate in patients with acute lung injury and a high negative rate in healthy individuals or patients with chronic progressive interstitial pneumonia or other respiratory diseases is set as the cutoff value I can do it.
- a suitable cutoff value for determining whether a subject suffers from acute lung injury using peripheral blood serum as a biological sample includes a value between 100 and 1500 pg / ml, Preferably a value between 200-1300 pg / ml, more preferably a value between 300-1200 pg / ml, even more preferably a value between 400-1000 pg / ml, most preferably between 500-900 pg / ml. Value.
- a suitable cutoff value for distinguishing acute lung injury from chronic progressive interstitial pneumonia using peripheral blood serum as a biological sample is a value between 100 and 1500 pg / ml.
- the method (1) of the present invention it is possible to differentiate between acute lung injury and the above-mentioned diseases that have clinical findings common to acute lung injury.
- the clinical findings of acute lung injury are similar to those of the above diseases, there is no factor that promotes collagen synthesis in the above diseases that have common clinical findings with acute lung injury. There is no increase.
- a biological sample eg, serum
- negative control that recognizes common clinical findings with acute lung injury and a patient with acute lung injury
- positive control compare the amount of HSP47 in the biological sample collected from the subject with that of the positive and negative controls.
- a correlation diagram between the amount of HSP47 in a specific biological sample (eg, serum) and the prevalence of acute lung injury is prepared in advance, and the amount of HSP47 in a biological sample collected from a subject is compared with the correlation diagram. May be.
- the comparison of the amount of HSP47 is preferably performed based on the presence or absence of a significant difference.
- the subject may suffer from acute lung injury rather than the above-mentioned disease that recognizes clinical findings in common with acute lung injury. Can be determined to be relatively high. Conversely, if the amount of HSP47 in a subject-derived biological sample is relatively low, the subject may be suffering from the above-mentioned disease that is not acute lung injury but has common clinical findings with acute lung injury It can be determined that the property is high.
- the present invention also provides a diagnostic agent for acute lung injury comprising an antibody that specifically recognizes the aforementioned HSP47.
- the diagnostic agent can be a diagnostic kit for acute lung injury.
- the diagnostic agent of the present invention is used, the subject easily suffers from acute lung injury by quantifying HSP47 in a biological sample derived from the subject by an immunological technique in the method (1) of the present invention described above. It is possible to determine whether or not acute lung injury and chronic progressive interstitial pneumonia or the above-mentioned diseases that have clinical findings in common with acute lung injury.
- the antibody may be directly or indirectly labeled with a labeling substance.
- Labeling substances include fluorescent substances (eg, FITC, rhodamine), radioactive substances (eg, 14 C, 3 H, 125 I), enzymes (eg, alkaline phosphatase, peroxidase), colored particles (eg, metal colloid particles, colored) Latex), biotin and the like.
- the antibody can be used in the form of an aqueous solution, but is preferably bound to a solid phase.
- solid phases include plates (eg, microwell plates), tubes, beads (eg, plastic beads, magnetic beads), chromatographic carriers (eg, Sepharose TM), membranes (eg, nitrocellulose membrane) , PVDF film), gel (eg, polyacrylamide gel), metal film (eg, gold film) and the like.
- the diagnostic agent of the present invention may contain reagents used for quantifying the amount of HSP47 in a biological sample, and these reagents and the like may contain HSP47 in advance. It may be combined with an antibody that specifically recognizes, or may be stored in a separate container. Reagents include buffers for diluting treatment solutions and antibodies, secondary antibodies, labeling substances (eg, fluorescent dyes, enzymes), reaction vessels, positive controls (eg, recombinant HSP47), negative controls, solid phases And instructions describing the test protocol. These elements can be mixed in advance if necessary.
- HSP47 in the method of the present invention may be quantified instead of or in addition to “an antibody that specifically recognizes HSP47”.
- a partial fragment thereof having a specific binding property to collagen or HSP47 may be used.
- the quantification of HSP47 by the technique using the partial fragment having specific binding property to collagen or HSP47 includes, for example, the following steps: (1 ′) A biological sample derived from a subject and a partial fragment thereof having specific binding properties with collagen or HSP47, and the portion having specific binding properties between HSP47 and collagen or HSP47 in the biological sample Forming a complex with the fragment; (2 ′) a step of detecting the complex formed in the step (1); and (3 ′) a step of calculating the amount of HSP47 in the biological sample from the amount of the complex detected in the step (2). .
- a biological sample derived from a subject is contacted with a partial fragment thereof having a specific binding property to collagen or HSP47, and the specific binding property between HSP47 and collagen or HSP47 in the biological sample is determined.
- a step of forming a complex with the partial fragment is determined.
- the collagen used in the method of the present invention is mammalian collagen.
- mammals include, for example, laboratory animals such as rodents and rabbits such as mice, rats, hamsters, and guinea pigs, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, humans, Examples include, but are not limited to, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees.
- the collagen used in the method of the present invention is preferably mammalian collagen of the same species as the subject. For example, when the subject is a human, preferably human collagen is used.
- human collagen means that the amino acid sequence of collagen has the same amino acid sequence as the amino acid sequence of collagen that is naturally expressed in humans.
- the collagen used in the method of the present invention may be any type of collagen as long as it has a property of specifically binding to HSP47.
- Mammalian collagen includes 27 types of type I to XXVII. Mammalian collagen is preferably type IV to V, more preferably type IV. It is known that at least type I to V mammalian collagen binds to HSP47 (Natsume T. et al., J. Biol. Chem., 269, 31224-31228, 1994).
- Collagen is a triple helix that usually contains three polypeptides.
- type I collagen is a triple that includes two ⁇ 1 chains (type I) and one ⁇ 2 chain (type I).
- a representative amino acid sequence of the ⁇ 1 chain of human type I collagen is shown in SEQ ID NO: 4, and a representative amino acid sequence of the ⁇ 2 chain of human type I collagen is shown in SEQ ID NO: 6, respectively.
- the “partial fragment of collagen having specific binding property to HSP47” may be a partial fragment of any region of collagen as long as it has specific binding property to HSP47.
- Collagen partial fragments having specific binding properties to HSP47 are usually three partial peptides (when the three polypeptides constituting collagen are defined as polypeptides A, B and C, each partial peptide is A polypeptide A fragment, a polypeptide B fragment, and a polypeptide C fragment).
- the size of the three partial peptides contained in the “partial fragment of collagen having specific binding to HSP47” is usually 9 amino acids or more, preferably 12 amino acids or more, more preferably 15 amino acids or more, and even more preferably 18 amino acids or more.
- the three partial peptides included in the “partial fragment of collagen having specific binding to HSP47” are each Gly-Xaa-Yaa (in the formula, Xaa and Yaa are included in the polypeptide constituting collagen). For example, 4 times or more, preferably 5 times or more, more preferably 6 times or more.
- the partial fragment preferably contains at least one arginine residue at the Yaa site (Koide, T. et al., J. Biol. Chem., 281, 3432-3438, 2006).
- At least one partial peptide contained in the “partial fragment of collagen having specific binding to HSP47” is at least one Yaa ⁇ 3 -Gly-Xaa ⁇ Amino acid sequence represented by 1- Arg-Gly (wherein Xaa- 1 is an arbitrary amino acid (preferably Pro), Yaa- 3 is an arbitrary amino acid (preferably Thr, Pro, Ser, Hyp, Val, Ala, Ile, Leu, Asn, Met, His, Phe or Tyr, more preferably Thr, Pro, Ser, Hyp, Val or Ala, more preferably Thr or Pro, most preferably Thr.
- Yaa -3 -Gly-Xaa -1 -Arg-Gly is Thr-Gly-Pro-Arg-Gly (Koide T. et al., J. Biol. Chem. 281, 11177-11185, 2006).
- any amino acid '' includes Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp , Tyr and Hyp are included.
- one or two or more are included in collagen or a partial fragment thereof having a specific binding property to HSP47 used in the quantification of HSP47.
- Such amino acid addition is permissible as long as collagen or a partial fragment thereof having a specific binding property to HSP47 specifically recognizes HSP47.
- the amino acid sequence to be added is not particularly limited, and examples thereof include tags for facilitating detection and purification of fast methionine, cysteine, and polypeptide.
- Tags include Flag tag, histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (eg GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tag, etc. It can be illustrated.
- the position where the amino acid sequence is added is preferably the N-terminus or C-terminus of the polypeptide.
- the partial fragment having specific binding property to collagen or HSP47 used in the present invention is isolated or purified. “Isolated or purified” means that an operation for removing components other than the target component from a natural state has been performed.
- the purity of the isolated or purified collagen or its partial fragment with specific binding to HSP47 is usually 50% or more, preferably 70% or more, more preferably 90% or more, and most preferably 95% or more (for example, substantially 100%).
- Collagen used in the method of the present invention or a partial fragment thereof having specific binding property to HSP47 has amino groups of amino acid residues at the N-terminal and C-terminal as protecting groups (for example, C1-6 such as formyl group, acetyl group, It may be protected with a C1-6 acyl group such as alkanoyl).
- protecting groups for example, C1-6 such as formyl group, acetyl group, It may be protected with a C1-6 acyl group such as alkanoyl).
- the collagen or a partial fragment thereof having a specific binding property to HSP47 may be directly or indirectly labeled with a labeling substance.
- Labeling substances include fluorescent substances (eg, FITC, rhodamine), radioactive substances (eg, 14 C, 3 H, 125 I), enzymes (eg, alkaline phosphatase, peroxidase), colored particles (eg, metal colloid particles, colored) Latex), biotin and the like.
- the recombinant collagen can be prepared, for example, by the following method.
- a polynucleotide encoding an amino acid sequence of a collagen subunit (for example, in the case of human Type 1 collagen, nucleotide sequences represented by SEQ ID NO: 3 and SEQ ID NO: 5 (GeneBank accession numbers: NM_000088.3 and NM_000089.3))
- a polynucleotide comprising a suitable expression vector inserted into a suitable host, transformed, associated with a collagen subunit molecule in the host, and subjected to desired recombination from the culture supernatant of the transformant.
- Collagen can be obtained.
- the host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, Bacillus subtilis, yeast, plant or animal cells can be used.
- collagen may be isolated or purified from a natural cell producing the same or a culture supernatant thereof by a known protein separation and purification technique. .
- it may be a protein synthesized chemically or biochemically by a cell-free translation system.
- partial fragment having specific binding property to collagen or HSP47 can be used in the form of an aqueous solution, but is preferably bound to a solid phase.
- solid phases include plates (eg, microwell plates), tubes, beads (eg, plastic beads, magnetic beads), chromatographic carriers (eg, Sepharose TM), membranes (eg, nitrocellulose membrane) , PVDF film), gel (eg, polyacrylamide gel), metal film (eg, gold film) and the like.
- plates, beads, membranes, and metal films are preferably used, and plates are most preferably used because of easy handling.
- the bond include covalent bond, ionic bond, physical adsorption, and the like, and are not particularly limited.
- the solid phase may be directly bonded to the solid phase or indirectly bonded to the solid phase using a substance known per se. Furthermore, in order to suppress non-specific adsorption and non-specific reaction, a phosphate buffer solution such as bovine serum albumin (BSA) or bovine milk protein is brought into contact with the solid phase, and the solid surface portion not coated with the antibody is removed. Blocking with the BSA or bovine milk protein is generally performed.
- BSA bovine serum albumin
- bovine milk protein Blocking with the BSA or bovine milk protein is generally performed.
- “collagen or a partial fragment thereof having a specific binding property to HSP47” and “HSP47” contained in the biological sample derived from the subject are contacted with each other in the reaction container.
- the embodiment, the order, the specific method, and the like are not particularly limited as long as they can be mixed with a partial fragment thereof having a specific binding property to HSP47 to interact with each other.
- the contact is made, for example, by adding the biological sample (extract thereof) to a plate on which “partial fragment having specific binding property to collagen or HSP47” is immobilized.
- the time for maintaining such contact is sufficient to form a complex by binding the collagen or a partial fragment thereof having specific binding property to HSP47 and HSP47 contained in the biological sample derived from the subject.
- the time is not particularly limited, but is usually from a few seconds to a few dozen hours, preferably from 1 minute to 2 hours, and most preferably from the viewpoint of promptly determining whether acute lung injury has occurred.
- the temperature condition for the contact is usually 4 ° C. to 50 ° C., preferably 4 ° C. to 37 ° C., and most preferably room temperature of about 15 ° C. to 30 ° C.
- the pH condition for carrying out the reaction is preferably 5.0 to 9.0, particularly preferably in the neutral range of 6.0 to 8.0.
- Step (2 ') is a step of determining whether or not HSP47 is present in the biological sample by detecting the complex formed in the step (1').
- the above detection is performed by detecting “HSP47” contained in the complex.
- This detection includes enzyme immunoassay (EIA), immunochromatography, latex agglutination, radioimmunoassay (RIA), fluorescence immunoassay (FIA), luminescence immunoassay, surface plasmon resonance ( SPR method) can be used.
- EIA enzyme immunoassay
- RIA radioimmunoassay
- FIA fluorescence immunoassay
- SPR method surface plasmon resonance
- the EIA method, immunochromatography method, FIA method and SPR method are preferable from the viewpoint of ease of operation and rapidity.
- the EIA method is preferably an ELISA method.
- An ELISA method using an enzyme-labeled antibody is applicable as a kind of ELISA method.
- HSP47 in blood is captured with any solid-phased “collagen or a partial fragment thereof having a specific binding property to HSP47”, and the captured HSP47 and enzyme-labeled “HSP47 specific Antigen-antibody reaction with the “recognizing antibody”.
- the time required for such a reaction is preferably 1 minute to 2 hours, more preferably 2 minutes to 30 minutes, from the viewpoint that rapid measurement is required.
- HSP47 is detected by detecting this enzyme.
- the enzyme-labeled antibody can be produced by binding (labeling) an enzyme and “an antibody that specifically recognizes HSP47” by a method known per se, such as the glutaraldehyde method or the maleimide method.
- Examples of the “enzyme” in the enzyme-labeled antibody include peroxidase, alkaline phosphatase, glucose oxidase, ⁇ -galactosidase and the like.
- an appropriate one is selected according to the selected labeling enzyme. For example, when peroxidase is selected as the enzyme, o-phenylenediamine (OPD), tetramethylbenzidine (TMB) or the like is used. When alkaline phosphatase is selected, p-nitrophenyl phosphate (PNPP) or the like is used. used. As the reaction stop solution and substrate solution, conventionally known ones can be appropriately used without particular limitation depending on the selected enzyme.
- blood collected from a subject is shaken at room temperature for a certain period of time, and then centrifuged to obtain serum.
- this serum is dispensed to a microplate on which any “partial fragment having specific binding ability to collagen or HSP47” is immobilized, and left at room temperature for a certain period of time.
- an antibody solution specifically recognizing HSP47 labeled with peroxidase or the like is dispensed onto the plate and reacted at room temperature for a certain period of time.
- the complex is detected by reacting with a chromogenic substrate solution such as TMB.
- step (3 ′) the amount of HSP47 in the biological sample is calculated from the amount of the complex detected in step (2 ′).
- the amount (concentration) of the complex detected in the step (2 ') can be measured by applying a signal such as fluorescence intensity or absorbance to a separately prepared calibration curve.
- concentration of HSP47 in the subject's biological sample can be quantified by multiplying the concentration obtained from the calibration curve by the dilution factor.
- the calibration curve shows the concentration of the sample obtained by serial dilution of the standard HSP47 protein preparation as the horizontal axis (vertical axis), and the vertical axis (horizontal axis) showing the signal of the antibody bound to HSP47 in the standard HSP47 protein preparation in arbitrary units.
- the measured value is plotted as an axis and expressed in an approximate expression.
- the calibration curve can be approximately expressed as a straight line by logarithmically displaying both axes.
- a correlation is made between the amount of HSP47 in a biological sample from the subject and the likelihood of suffering from acute lung injury, and whether or not the subject is suffering from acute lung injury based on the correlation. judge.
- the amount of HSP47 in the biological sample is higher in patients with acute lung injury than in healthy subjects and patients with chronic progressive interstitial pneumonia.
- the above determination is made based on a positive correlation between the amount of HSP47 in a biological sample derived from such a subject and the likelihood of suffering from acute lung injury.
- a biological sample eg, serum
- a biological sample eg, serum
- negative control chronic progressive interstitial pneumonia
- positive control positive control
- HSP47 in the biological sample collected from the subject Is compared to that of the positive and negative controls.
- a correlation diagram between the amount of HSP47 in a specific biological sample (eg, serum) and the prevalence of acute lung injury is prepared in advance, and the amount of HSP47 in a biological sample collected from a subject is compared with the correlation diagram. May be.
- the comparison of the amount of HSP47 is preferably performed based on the presence or absence of a significant difference.
- the method (2) of the present invention it is possible to distinguish between acute lung injury and the above-mentioned diseases that have clinical findings common to acute lung injury.
- the clinical findings of acute lung injury are similar to those of the above diseases, there is no factor that promotes collagen synthesis in the above diseases that have common clinical findings with acute lung injury. There is no increase.
- a biological sample eg, serum
- negative control that recognizes common clinical findings with acute lung injury and a patient with acute lung injury
- positive control compare the amount of HSP47 in the biological sample collected from the subject with that of the positive and negative controls.
- a correlation diagram between the amount of HSP47 in a specific biological sample (eg, serum) and the prevalence of acute lung injury is prepared in advance, and the amount of HSP47 in a biological sample collected from a subject is compared with the correlation diagram. May be.
- the comparison of the amount of HSP47 is preferably performed based on the presence or absence of a significant difference.
- the subject may suffer from acute lung injury rather than the above-mentioned disease that recognizes clinical findings in common with acute lung injury. Can be determined to be relatively high. Conversely, if the amount of HSP47 in a subject-derived biological sample is relatively low, the subject may be suffering from the above-mentioned disease that is not acute lung injury but has common clinical findings with acute lung injury It can be determined that the property is high.
- the present invention also provides a diagnostic agent for acute lung injury comprising the above-described collagen or a partial fragment thereof having a specific binding property to HSP47.
- the diagnostic agent can be a diagnostic kit for acute lung injury.
- the diagnostic agent of the present invention is used, the subject easily suffers from acute lung injury by quantifying HSP47 in a biological sample derived from the subject by immunological technique in the above-described method (2) of the present invention. It is possible to determine whether or not acute lung injury and chronic progressive interstitial pneumonia or the above-mentioned diseases that have clinical findings in common with acute lung injury.
- the partial fragment having specific binding properties to collagen or HSP47 can be used in the state of an aqueous solution to which nothing else is bound, but it is preferably bound to a solid phase.
- solid phases include plates (eg, microwell plates), tubes, beads (eg, plastic beads, magnetic beads), chromatographic carriers (eg, Sepharose TM), membranes (eg, nitrocellulose membrane) , PVDF film), gel (eg, polyacrylamide gel), metal film (eg, gold film) and the like.
- the diagnostic agent of the present invention may contain reagents used for quantifying the amount of HSP47 in a biological sample in addition to collagen or a partial fragment thereof having specific binding properties to HSP47, and these reagents Or the like may be preliminarily combined with collagen or a partial fragment thereof having a specific binding property to HSP47, or may be stored in a separate container.
- reagents include treatment solutions and buffers for diluting antibodies, antibodies that specifically recognize HSP47 derived from subjects, labeling substances (eg, fluorescent dyes, enzymes), reaction vessels, positive controls (eg, combinations) HSP47), negative control, solid phase, and instructions describing the test protocol. These elements can be mixed in advance if necessary.
- heat shock protein 47 as a biomarker for predicting the onset of acute lung injury in the hyperacute phase before meeting the clinical definition of acute lung injury
- acute lung injury The onset of acute lung injury can be predicted by quantifying heat shock protein 47 in a biological sample from a subject in the hyperacute phase before meeting the clinical definition of
- a “subject” can be a subject that does not meet the clinical definition of acute lung injury.
- the subject include, but are not limited to, healthy subjects, subjects suffering from interstitial pneumonia, subjects suffering from acute respiratory diseases such as bacterial pneumonia and swallowing pneumonia .
- the subject can be a subject that has not shown clinical findings of acute lung injury.
- Quantification of the heat shock protein 47 in the biological sample derived from the subject can be performed in the same manner as the quantification in the method (1) of the present invention and the method (2) of the present invention.
- Correlation between the amount of HSP47 in the biological sample derived from the subject and the possibility of developing acute lung injury is performed, and whether or not the subject develops acute lung injury is determined based on the correlation.
- the higher the amount of HSP47 in the biological sample the higher the possibility of developing acute lung injury.
- the determination is made based on a positive correlation between the amount of HSP47 in such a subject-derived biological sample and the likelihood of developing acute lung injury.
- the above determination is made, for example, by previously creating a correlation diagram between the amount of HSP47 in a specific biological sample (eg, serum) and the possibility of developing acute lung injury, and the amount of HSP47 in a biological sample collected from a subject. May be compared with its correlation diagram.
- a specific biological sample eg, serum
- the amount of HSP47 in a biological sample collected from a subject May be compared with its correlation diagram.
- Serum HSP47 rises during follow-up of patients with interstitial pneumonia and is likely to develop rapidly progressive interstitial pneumonia that develops acute exacerbations in a short span and meets the clinical definition of acute lung injury It can be judged. Since acute exacerbation of interstitial pneumonia presents only a common cold-like symptom in the early stage of onset, it is often treated as a mere cold and becomes severe and fatal in the meantime. Even if an acute exacerbation of interstitial pneumonia is suspected, it is difficult to make an early diagnosis because hypoxemia and abnormal shadows on chest x-rays do not appear unless the disease becomes severe to some extent.
- heat shock protein 47 as a biomarker for grasping activity of acute lung injury and predicting prognosis
- a biological sample derived from a subject suffering from acute lung injury By quantifying the heat shock protein 47, the activity of acute lung injury can be grasped and the prognosis can be predicted.
- Serum HSP47 levels in subjects suffering from acute lung injury correlate with disease activity of acute lung injury, so by measuring serum HSP47, activity and severity can be ascertained, Treatment effect judgment and prognosis prediction can be performed.
- Quantification of heat shock protein 47 in a biological sample derived from a subject suffering from acute lung injury can be performed in the same manner as the quantification in the method (1) of the present invention and the method (2) of the present invention.
- Correlation between the amount of HSP47 in a biological sample from a subject suffering from acute lung injury and the disease activity of acute lung injury, and based on the correlation, the activity of acute lung injury in the subject Determine the severity.
- the determination is made, for example, by creating a correlation diagram between the amount of HSP47 in a specific biological sample (eg, serum) and the disease activity of acute lung injury in advance, and calculating the amount of HSP47 in the biological sample collected from the subject. The comparison is made with the correlation diagram.
- a specific biological sample eg, serum
- ALI 32 cases
- COP 12 cases
- I-UIP idiopathic UIP
- I-NSIP idiopathic NSIP
- CVD -UIP 11 cases
- CVD-NSIP idiopathic NSIP
- Volunteer healthy individuals 18 cases.
- Cases of ALI include rapidly progressive interstitial pneumonia that meets the clinical definition of ALI.
- the test protocol is approved by the institutional review board, and informed consent is obtained from the subjects. Further specific clinical background is shown in Table 1.
- P / F ratio, A-aDO 2 and Respiratory index are PaO 2 / FiO 2 (mmHg) (arterial oxygen partial pressure divided by inhaled oxygen partial pressure), alveolar air artery blood oxygen Partial pressure difference (difference between alveolar oxygen partial pressure (PAO 2 ) and arterial oxygen partial pressure (PaO 2 )) and A-aDO 2 / PaO 2 (alveolar air arterial oxygen partial pressure difference divided by arterial oxygen partial pressure) ),
- KL-6, SP-D, SP-A and LDH indicate serum marker values.
- P / F ratio, A-aDO 2 and respiratory index are statistically significant at p ⁇ 0.01 when compared between ALI patients and COP, idiopathic UIP, idiopathic NSIP, CVD-UIP, and CVD-NSIP patients, respectively. There was a difference.
- Example 1 Preparation of HSP47 protein Preparation of collagen-immobilized column Suspend CNBr-activated Sepharose 4B (GE Bioscience) in 1 mM HCl and mix with an equal volume of 3 mg / ml porcine type I collagen (Nitta gelatin) in 1 mM HCl . Two volumes of 0.2 M NaHCO 3 and 0.5 M NaCl were added, and the mixture was stirred at room temperature for 2 hours using a rotator. The resin is collected by centrifugation, suspended in 1 M ethanolamine hydrochloride (pH 8.5), and stirred at 4 ° C for 1 hour.
- CNBr-activated Sepharose 4B GE Bioscience
- the resin was washed with 0.2 M NaHCO 3 , 0.5 M NaCl, and then with 0.1 M sodium acetate buffer (pH 4.0) containing 0.5 M sodium chloride. This was used as collagen-Sepharose 4B resin for HSP47 purification.
- Preparation of recombinant HSP47 protein The cDNA of human HSP47 was subcloned into pET3a to construct an expression plasmid (pET-hH47). Escherichia coli BL21 (DE3) was transformed with pET-hH47. The transformed strain was cultured at 25 ° C., 0.1 mM isopropyl thiogalactoside was added, and further cultured for 2 hours.
- Example 2 Preparation of antibody-immobilized plate Rabbit anti-HSP47 polyclonal antibody (Santa Cruz Biotechnology) was diluted to 1.0 ⁇ g / mL with 50 mM sodium carbonate buffer (pH 9.6) in a 96-well microplate (Nunk). One was added at 50 ⁇ L / well and allowed to stand overnight at 4 ° C. to allow the antibody to bind to the plate surface. After discarding the antigen solution, add 2% BSA (Sigma) -containing phosphate buffer at 200 ⁇ L / well to suppress non-specific adsorption to the non-specific plate and non-specific reaction. The plate was blocked by allowing to stand. Subsequently, the well was washed three times with PBS containing 0.05% Tween 80 (PBST) to prepare an anti-HSP47 antibody-immobilized plate.
- PBST PBS containing 0.05% Tween 80
- Recombinant HSP47 used the sample purified by the above method.
- the recombinant HSP47 solution (in 1% BSA-PBST, pH 7.4) is added to the plate prepared in (1) at a concentration of 100 ⁇ L / well, and left at room temperature for 2 hours to obtain anti-HSP47 antibody.
- a complex bound to recombinant HSP47 was formed.
- mouse anti-HSP47 monoclonal antibody (0.5 ⁇ g / mL, manufactured by Stressgen) was added at 100 ⁇ L / well, and the mixture was allowed to stand at 37 ° C.
- HSP47 monoclonal antibody was conjugated.
- POD-labeled anti-mouse IgG antibody diluted 10,000 times, manufactured by Biosource
- Biosource 3,3 ′, 5,5′-tetramethylbenzidine substrate solution
- Example 3 Detection of HSP47 using human serum as a specimen
- the serum of a patient suffering from acute lung injury or various chronic progressive interstitial pneumonia was used as a specimen instead of the recombinant HSP47 solution, and the same as in Example 2
- the operation was performed and the absorbance was measured.
- the HSP47 concentration in the serum of each patient was calculated. The results are shown in FIG.
- HSP47 levels were low in patients with chronic progressive interstitial pneumonia (COP, I-UIP, I-NSIP, CVD-UIP, CVD-NSIP) and in healthy individuals, but very high in patients with acute lung injury A high level of concentration was shown (FIG. 1).
- COP chronic progressive interstitial pneumonia
- I-UIP I-NSIP
- CVD-UIP CVD-NSIP
- CVD-NSIP chronic progressive interstitial pneumonia
- ROC Receiver Operating Characteristic
- Serum HSP47 protein ROC curve is more complete in acute lung injury and chronic progressive interstitial pneumonia compared to KL-6, SP-D, SP-A and LDH ROC curves It was distributed at the position closest to the upper left corner that can be separated into two.
- HSP47 is a much more useful marker than the conventional serum markers KL-6, SP-D, SP-A, and LDH.
- the mortality rate after 1 month of ALI was 31.3% (10 cases / 32 cases), and the mortality rate after 3 months was 50% (16 cases / 32 cases).
- pneumonia COP, I-UIP, I-NSIP, CVD-UIP, CVD-NSIP
- Example 5 Furthermore, the number of subject subjects was increased and the test was conducted.
- Study population for serum HSP47 The subjects in this study consisted of 116 patients admitted to Nagasaki University Hospital from April 1991 to April 2011 and 18 healthy adult volunteers. Patients included 47 patients with rapid progressive interstitial pneumonia (RPIP) included in acute lung injury of the present invention, 12 patients with idiopathic organized pneumonia (COP), 19 patients with idiopathic UIP, idiopathic We included 16 patients with sex NSIP, 11 patients with CVD-UIP, and 11 patients with CVD-NSIP.
- RPIP rapid progressive interstitial pneumonia
- COP idiopathic organized pneumonia
- idiopathic idiopathic
- Patient characteristics Table 2 shows the patient characteristics collected in this study.
- the P / F ratio was significantly lower than that in the COP, idiopathic UIP, idiopathic NSIP, CVD-UIP and CVD-NSIP groups (P ⁇ 0.01).
- the alveolar arterial partial pressure difference (Aa DO 2 ) was significantly lower than that in the COP, idiopathic UIP, idiopathic NSIP, CVD-UIP and CVD-NSIP groups (P ⁇ 0.01).
- the APACHE (Acute Physiology and Chronic Health Evaluation) II score and SOFA (Sequential Organ Failure Assessment) score in the RPIP group were 15.0 (range 8 to 35) and 4.0 (range 2 to 12), respectively.
- Serum levels of HSP47, KL-6, SP-A, SP-D and LDH were quantified. Serum levels of HSP47 in patients with RPIP (median 1530.2 [range 631.2 to 14589.9] pg / ml) were COP (239.1 [16.6 to 476.6] pg / ml) (P ⁇ 0.01), idiopathic UIP (330.9 [ 105.1-487.6] pg / ml) (P ⁇ 0.01), idiopathic NSIP (290.7 [24.8-603.0] pg / ml) (P ⁇ 0.01), CVD-UIP (348.8 [122.8-602.0] pg / ml) (P ⁇ 0.05), CVD-NSIP (383.1 [101.9-721.4] pg / ml) (P ⁇ 0.05), and healthy volunteers (547.9 [332.1-879.
- Serum levels of KL-6 in patients with idiopathic UIP, idiopathic NSIP, and RPIP patients with idiopathic UIP (1460.0 [444-4340] U / ml) (P ⁇ 0.05), patients with idiopathic NSIP (1568.5 [192-4745] U / ml) (P ⁇ 0.05) and RPIP patients (918.5 [107-5800] U / ml) (P ⁇ 0.05) were healthy volunteers (195.0 [144-322] U / ml). It was significantly higher than the level of ml).
- Serum levels of SP-A in patients with RPIP and idiopathic UIP was significantly higher than that of healthy volunteers (22.8 [12.1-60.8] ng / ml).
- COP, idiopathic UIP, idiopathic NSIP, CVD-UIP, CVD-NSIP, RPIP patients (COP patients (105.7 [27.8-247.0] ng / ml), idiopathic UIP patients (316.0 [93.1-721.0] ng) / ml), idiopathic NSIP patients (477.0 [17.2 to 942.0] ng / ml), CVD-UIP patients (187.0 [33.5 to 264.0] ng / ml), CVD-NSIP patients (111.5 [33.2 to 275.0] ng / ml), RPIP patients (280.5 [29.0 to 450.0] ng / ml)), and healthy volunteers (17.3 [17.3 to 53.3] ng / ml), there is no significant difference in serum levels of SP-D There was no ( Figure 10).
- Receiver operating characteristic curve A receiver operating characteristic curve was prepared in the same manner as in Example 4 (FIG. 12). Based on the receiver operating characteristic curve, the cut-off value for HSP47 yielding the most accurate diagnosis was 617.1 pg / ml.
- the cutoff value 617.1 pg / ml for HSP47 is RPIP and other interstitial pneumonia (COP, idiopathic UIP, idiopathic NSIP, CVD-UIP, and CVD-NSIP with 100% sensitivity and 98.6% specificity. ).
- the diagnostic accuracy was 99.1%.
- Use of serum HSP47 resulted in the largest area under the curve.
- the area under the curve for the diagnosis of rapidly progressive interstitial pneumonia was 0.998.
- the area under the curve for the diagnosis of rapidly progressive interstitial pneumonia based on serum levels of KL-6, SP-A, SP-D and LDH was only 0.475, 0.728, 0.595 and 0.776, respectively. there were.
- HSP47 is a much more useful marker than the conventional serum markers KL-6, SP-D, SP-A, and LDH.
- the test method of the present invention can detect HSP47, which is a novel biomarker of acute lung injury, simply and quickly. Further, by using the diagnostic kit for acute lung injury and the diagnostic agent for acute lung injury of the present invention, whether or not the patient is suffering from acute lung injury can be diagnosed simply, quickly and with high accuracy.
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Abstract
Description
[1] 被験体由来の生体試料におけるヒートショックプロテイン47を定量することを含む、被験体が急性肺損傷に罹患しているか否かを判定するための方法。
[2] 急性肺損傷と慢性進行性間質性肺炎との判別方法である、[1]に記載の方法。
[3] 急性肺損傷と大動脈解離、急性心不全、慢性心不全急性増悪、びまん性肺胞出血、癌性リンパ管症、再膨張性肺水腫、過剰輸液による肺水腫、神経原性肺水腫、肺結核、又は粟粒結核との判別方法である、[1]に記載の方法。
[4] 生体試料が血液、血清又は血漿である、[1]~[3]のいずれかに記載の方法。
[5] ヒートショックプロテイン47を特異的に認識する抗体及び/又は、コラーゲン又はヒートショックプロテイン47との特異的結合性を有するその部分断片を含む、急性肺損傷の診断薬。
[6] 被験体由来の生体試料におけるヒートショックプロテイン47を定量することを含む、被験体が急性肺損傷を発症するか否かを判定するための方法。
[7] 被験体由来の生体試料におけるヒートショックプロテイン47を定量することを含む、被験体の急性肺損傷の治療効果を判定又は予後を予測するための方法。
本発明は、被験体由来の生体試料におけるヒートショックプロテイン47を定量することを含む、被験体が急性肺損傷に罹患しているか否かを判定するための方法(方法(1))を提供するものである。
(I)急性発症。
(II)低酸素血症(PaO2/FiO2が300 mmHg以下)。
(FiO2=吸気O2濃度。PaO2の単位はmmHg、FiO2は小数(例、0.5)。PaO2/FiO2が200 mmHg以下のケースが急性呼吸促迫症候群である。)
(III)胸部X線写真にて両側性の肺浸潤影を認める。
(IV)肺動脈楔入圧が18mmHg以下または理学的に左房圧上昇の臨床所見がない。
上記基準を全て満たすものが急性肺損傷と定義される。
(1)被験体由来の生体試料と、HSP47を特異的に認識する抗体とを接触させ、生体試料中のHSP47とHSP47を特異的に認識する抗体との複合体を形成させる工程;
(2)上記工程(1)で形成された複合体を検出する工程;及び
(3)上記工程(2)で検出された複合体の量から生体試料中のHSP47の量を算出する工程。
(1’)被験体由来の生体試料と、コラーゲン又はHSP47との特異的結合性を有するその部分断片とを接触させ、生体試料中のHSP47とコラーゲン又はHSP47との特異的結合性を有するその部分断片との複合体を形成させる工程;
(2’)上記工程(1)で形成された複合体を検出する工程;及び
(3’)上記工程(2)で検出された複合体の量から生体試料中のHSP47の量を算出する工程。
本発明の別の態様においては、急性肺損傷の臨床的定義を満たす以前の超急性期に、被験体由来の生体試料におけるヒートショックプロテイン47を定量することにより、急性肺損傷の発症を予測することができる。
本発明の別の態様においては、急性肺損傷に罹患している被験体由来の生体試料におけるヒートショックプロテイン47を定量することにより、急性肺損傷の活動性把握、予後予測をすることができる。
対象とした被験者は長崎大学病院の患者で、内訳はALI(32例)、COP(12例)、idiopathic UIP(I-UIP;19例)、idiopathic NSIP(I-NSIP;16例)、CVD-UIP(11例)、CVD-NSIP(12例)及び並びに有志の健常者(18例)である。なお、ALIの症例は、ALIの臨床的定義を満たす急速進行性間質性肺炎を含んでいる。試験プロトコールはinstitutional review boardにより承認され、被験者からはinformed consentが得られている。更に具体的な臨床背景を表1に示す。表中、P/F比、A-aDO2及び呼吸指数(Respiratory index)はそれぞれPaO2/FiO2(mmHg)(動脈血酸素分圧を吸入気酸素分圧で除したもの)、肺胞気動脈血酸素分圧較差(肺胞酸素分圧(PAO2)と動脈血酸素分圧(PaO2)の差)及びA-aDO2/PaO2(肺胞気動脈血酸素分圧較差を動脈血酸素分圧で除したもの)を示し、KL-6、SP-D、SP-A及びLDHは血清マーカーの値を示している。P/F比、A-aDO2及び呼吸指数は、ALIの患者とCOP、idiopathic UIP、idiopathic NSIP、CVD-UIP、CVD-NSIPの患者でそれぞれ比較した場合、統計学的にp<0.01で有意差を認めた。
HSP47タンパク質の調製
コラーゲン固定化カラムの作製
CNBr-activated Sepharose 4B (GE Bioscience)を1 mM HClに懸濁し、等量の3 mg/mlブタI型コラーゲン(新田ゼラチン)の1 mM HCl溶液と混ぜる。2倍量の0.2 M NaHCO3, 0.5 M NaClを添加し、室温で2時間ローテーターを用いて攪拌した。樹脂を遠心により回収し、1 M エタノールアミン塩酸 (pH8.5) に懸濁して4℃,1時間攪拌する。樹脂を0.2 M NaHCO3, 0.5 M NaCl、次いで0.5 M 塩化ナトリウム含有0.1 M 酢酸ナトリウム緩衝液(pH4.0)で洗浄した。これをHSP47精製用のコラーゲン-Sepharose 4B樹脂とした。
組換HSP47タンパク質の調製
ヒトHSP47のcDNAをpET3aにサブクローニングし、発現プラスミドを構築した(pET-hH47)。大腸菌BL21(DE3)にpET-hH47を形質変換した。形質変換株を25℃で培養し、0.1 mM isopropyl thiogalactosideを添加し、さらに2時間培養した。以下の精製操作は4℃で行った。組換大腸菌菌体を遠心で回収し、抽出バッファー(50 mM トリス塩酸, pH8.0, 150 mM 塩化ナトリウム, 5 mM EDTA, 1 mM フッ化フェニルメチルスルホニル, 1 μg/ml ペプスタチンA, 1 μg/ml ロイペプチン, 0.2% Nonidet P40, 30% グリセロール)に懸濁した。超音波破砕後、12,000 rpm, 15分遠心し、上清を150 mM 塩化ナトリウム含有50 mM トリス塩酸, pH8.0で平衡化したコラーゲン-Sepharose 4Bカラム(1 literの培養あたりbedvolume 360 ml, 流速 1 ml/min)に供した。カラムを50 mM トリス塩酸, pH8.0, 150 mM塩化ナトリウム, 1% Nonidet P40 でカラムを洗滌した。樹脂に結合したHSP47を、MES緩衝液, pH5.8, 150 mM NaCl, 1% Nonidet P40, 10% グリセロールからなる溶液で溶出した。10分の1量の1 M トリス塩酸, pH8.0 で中和、グリセロールを最終濃度20%となるように加えた。
抗体固相化プレートの作製
96ウェルマイクロプレート(ヌンク社製)に、ウサギ抗HSP47ポリクローナル抗体(Santa Cruz Biotechnology社製)を50 mM 炭酸ナトリウム緩衝液 (pH9.6) で1.0μg/mL で希釈したものを50μL/ウェルで加え、4℃で一晩静置してプレート表面に抗体を結合させた。抗原溶液を捨てた後、非特異的なプレートへの吸着や非特異的反応を抑制するために、2% BSA(シグマ社製)含有リン酸緩衝液を200μL/ウェルで加え、室温で2時間静置してプレートのブロッキングを行った。次いで0.05% Tween 80含有PBS (PBST)でウェルを3回洗浄して、抗HSP47抗体固相化プレートを作製した。
組換えHSP47は、上記の方法で精製した標品を用いた。(1)で作製したプレートに、前記組換えHSP47溶液(1% BSA-PBST中、pH7.4)を、濃度を変えて100μL/ウェルで加え、室温で2時間静置して抗HSP47抗体と組換えHSP47とが結合した複合体を形成させた。PBSTでウェルを3回洗浄後、マウス抗HSP47モノクローナル抗体(0.5 μg/mL,Stressgen社製)を100μL/ウェルで加え、37℃で2時間静置して複合体中の組換えHSP47にマウス抗HSP47モノクローナル抗体を結合させた。PBSTでウェルを3回洗浄後、POD標識した抗マウスIgG抗体(10,000倍希釈,Biosource社製)を100μL/ウェルで加え、37℃で2時間静置した。PBSTでウェルを3回洗浄後、3,3’,5,5’-テトラメチルベンジジン基質溶液(Biosource社製)を100μL/ウェルで加え、室温で15分静置して発色させた。等量の1Mリン酸で反応を停止させ、450nmの吸光度を測定した。得られたデータに基づいて、検量線を作成した。
ヒト血清を検体として用いた、HSP47の検出
組換えHSP47溶液の代わりに急性肺損傷又は種々の慢性進行性間質性肺炎に罹患している患者の血清を検体として用い、実施例2と同様の操作を行って吸光度を測定した。実施例2で作成した検量線に基づき、各患者の血清中のHSP47濃度を算出した。結果を図1に示す。
KL-6、SP-D、SP-A及びLDHについても、上記被験者の血清を用いて測定を行った。それぞれについて、図2~5に結果を示す。いずれの血清マーカーに関しても、慢性進行性間質性肺炎と急性肺損傷の患者間で有意差は認めなかった。
受信者動作特性曲線解析(Receiver Operating Characteristic (ROC) curve analysis)
被験者におけるHSP47蛋白、KL-6、SP-D、SP-A及びLDHの各血清マーカーの測定値に基づいて、受信者動作特性(ROC)曲線を作成した(作成方法は松尾収二、高橋浩:検査診断学におけるROC曲線の利用の実際.臨床病理 42:585-590, 1994を参照)。結果を図6に示す。
この検討では、ALIの1か月後死亡率は31.3 %(10例/32例)、3か月後の死亡率は50 %(16例/32例)であったが、慢性進行性間質性肺炎(COP、I-UIP、I-NSIP、CVD-UIP、CVD-NSIP)では1か月後、3か月後は全例生存していた。この結果は、HSP47が患者の予後予測因子として有用なマーカーであることを示している。
更に対象被験者数を増加させ、試験を行った。
本研究の被験者は、1991年4月~2011年4月に長崎大学病院に入院した116人の患者と18人の成人健常者有志で構成した。患者には、本発明の急性肺損傷に含まれる急速進行性間質性肺炎(RPIP)の患者47人、特発性器質化肺炎(COP)の患者12人、特発性UIPの患者19人、特発性NSIPの患者16人、CVD-UIPの患者11人、CVD-NSIPの患者11人を含めた。以下の基準:1)30日以内の急速な経過で呼吸状態が悪化;2)高解像度胸部CTスキャンにて新たな両側性すりガラス状陰影及び/又は浸潤影を認める;3)安静時PaO2/吸気酸素分画(FiO2)比(P/F比)<300mmHg;及び4)明白な感染、気胸、肺血栓塞栓症、心不全又はそれ以外の急性肺損傷原因(身体的外傷、輸血又は毒物吸入)の欠如の4項目を満たす患者をRPIPと診断した。RPIPの患者は、IPFの急性増悪の患者17人、急性間質性肺炎(AIP)の患者10人、膠原血管病関連の間質性肺炎の患者8人及び薬剤性間質性肺炎の患者12人で構成した。
連続型変数の値は中央値(範囲)として表現した。群間の差は、適宜、連続型変数についての分散分析又はKruscal-Wallis検定、及びカテゴリー変数についてのχ2検定を用いて検証した。分散分析により有意差が見出された場合、Scheffe法を用いてペアワイズ比較を行った。受信者動作特性曲線の左上隅の座標点を用いて、RPIPと他の間質性肺炎(COP、特発性UIP、特発性NSIP、CVD-UIP及びCVD-NSIP)とを鑑別するための至適カットオフ値を決定した。統計解析は統計ソフトウェアパッケージ(SAS 9.1.3, SAS Institute, Cary, NC)を用いて行った。p value<0.05を統計的有意とみなした。
本研究において参集した患者の特性を表2に示す。RPIP群においては、COP、特発性UIP、特発性NSIP、CVD-UIP及びCVD-NSIP群と比較してP/F比が有意に低かった(P<0.01)。RPIP群においては、COP、特発性UIP、特発性NSIP、CVD-UIP及びCVD-NSIP群と比較して肺胞動脈分圧格差(A-a DO2)が有意に低かった(P<0.01)。RPIP群におけるAPACHE(Acute Physiology and Chronic Health Evaluation)IIスコア及びSOFA(Sequential Organ Failure Assessment)スコアはそれぞれ、15.0(範囲は8~35)及び4.0(範囲は2~12)であった。
RPIP群においては、30日死亡率は34.0%(患者47人のうち16人)で、90日死亡率は51.1%(患者47人のうち24人)であった。対照的に、COP、特発性UIP、特発性NSIP、CVD-UIP又はCVD-NSIP群の患者は、90日以内には誰も死亡しなかった。
実施例2と同様にして患者におけるHSP47の血清中レベルを定量した。RPIPの患者におけるHSP47の血清中レベル(中央値1530.2[範囲は631.2~14589.9]pg/ml)は、COP(239.1[16.6~476.6]pg/ml)(P<0.01)、特発性UIP(330.9[105.1~487.6]pg/ml)(P<0.01)、特発性NSIP(290.7[24.8~603.0]pg/ml)(P<0.01)、CVD-UIP(348.8[122.8~602.0]pg/ml)(P<0.05)、CVD-NSIP(383.1[101.9~721.4]pg/ml)(P<0.05)、及び健常者の有志(547.9[332.1~879.8]pg/ml)(P<0.05)よりも有意に高かった。COP、特発性UIP、特発性NSIP、CVD-UIP、CVD-NSIPの患者、及び健常者の有志の中では、HSP47の血清中レベルには有意な差がなかった(図7)。
受信者動作特性曲線
実施例4と同様にして受信者動作特性曲線を作成した(図12)。当該受信者動作特性曲線に基づくと、最も高精度な診断をもたらす、HSP47についてのカットオフ値は617.1pg/mlであった。HSP47についてのカットオフ値617.1pg/mlは、100%の感度及び98.6%の特異度でRPIPと他の間質性肺炎(COP、特発性UIP、特発性NSIP、CVD-UIP、及びCVD-NSIP)とを鑑別した。診断精度は99.1%であった。血清中HSP47の使用により曲線の下で最大面積がもたらされた。HSP47の血清中レベルを用いると、急速進行性間質性肺炎の診断についての曲線下面積は0.998であった。対照的に、KL-6、SP-A、SP-D及びLDHの血清中レベルに基づく急速進行性間質性肺炎の診断についての曲線下の面積は、それぞれたった0.475、0.728、0.595及び0.776であった。
Claims (7)
- 被験体由来の生体試料におけるヒートショックプロテイン47を定量することを含む、被験体が急性肺損傷に罹患しているか否かを判定するための方法。
- 急性肺損傷と慢性進行性間質性肺炎との判別方法である、請求項1に記載の方法。
- 急性肺損傷と大動脈解離、急性心不全、慢性心不全急性増悪、びまん性肺胞出血、癌性リンパ管症、再膨張性肺水腫、過剰輸液による肺水腫、神経原性肺水腫、肺結核、又は粟粒結核との判別方法である、請求項1に記載の方法。
- 生体試料が血液、血清又は血漿である、請求項1~3のいずれか1項に記載の方法。
- ヒートショックプロテイン47を特異的に認識する抗体及び/又は、コラーゲン又はヒートショックプロテイン47との特異的結合性を有するその部分断片を含む、急性肺損傷の診断薬。
- 被験体由来の生体試料におけるヒートショックプロテイン47を定量することを含む、被験体が急性肺損傷を発症するか否かを判定するための方法。
- 被験体由来の生体試料におけるヒートショックプロテイン47を定量することを含む、被験体の急性肺損傷の治療効果を判定又は予後を予測するための方法。
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US13/984,623 US20130323763A1 (en) | 2011-02-10 | 2012-02-10 | Method for diagnosing acute lung injury |
EP12744724.1A EP2674755B1 (en) | 2011-02-10 | 2012-02-10 | Method for diagnosing acute lung injury |
JP2012556945A JP5966227B2 (ja) | 2011-02-10 | 2012-02-10 | 急性肺損傷診断方法 |
CN201280014503.3A CN103430026B (zh) | 2011-02-10 | 2012-02-10 | 急性肺损伤的诊断方法 |
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EP (1) | EP2674755B1 (ja) |
JP (1) | JP5966227B2 (ja) |
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Cited By (2)
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KR101468047B1 (ko) * | 2013-01-16 | 2014-12-02 | 고려대학교 산학협력단 | 열충격단백질군의 mRNA 발현 양상 분석을 통한 사인 추정방법 |
JP2019116507A (ja) * | 2019-04-25 | 2019-07-18 | 有限会社オービット | Hsp47の発現促進剤、脱毛抑制方法、Hsp47の発現促進剤の製造方法及び飲食物の製造方法 |
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CA2774383A1 (en) | 2011-06-22 | 2012-12-22 | Shaf Keshavjee | Repaired organ and method for making the same |
CA3033650A1 (en) * | 2016-08-17 | 2018-02-22 | The Regents Of The University Of California | A novel immunoprobe-based method to assess organ injury status through a biofluid-based cell-free dna (cfdna) assay |
CN112710847B (zh) * | 2020-12-17 | 2022-04-08 | 温州医科大学慈溪生物医药研究院 | Apelin蛋白在制备诊断呼吸系统疾病试剂的应用 |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101468047B1 (ko) * | 2013-01-16 | 2014-12-02 | 고려대학교 산학협력단 | 열충격단백질군의 mRNA 발현 양상 분석을 통한 사인 추정방법 |
JP2019116507A (ja) * | 2019-04-25 | 2019-07-18 | 有限会社オービット | Hsp47の発現促進剤、脱毛抑制方法、Hsp47の発現促進剤の製造方法及び飲食物の製造方法 |
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Publication number | Publication date |
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EP2674755A1 (en) | 2013-12-18 |
CN103430026A (zh) | 2013-12-04 |
JP5966227B2 (ja) | 2016-08-10 |
JPWO2012108538A1 (ja) | 2014-07-03 |
EP2674755B1 (en) | 2017-07-26 |
US20130323763A1 (en) | 2013-12-05 |
CN103430026B (zh) | 2016-01-27 |
EP2674755A4 (en) | 2014-12-03 |
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