JP6083784B2 - Method for detecting an exacerbation index of chronic obstructive pulmonary disease - Google Patents

Description

  The present invention relates to a method for detecting an exacerbation index that can be used in diagnosis of exacerbation of chronic obstructive pulmonary disease.

  Chronic obstructive pulmonary disease (COPD) is an inflammatory disease of the lungs caused by long-term inhalation exposure to harmful substances, mainly tobacco smoke. Patients with this disease exhibit airflow obstruction that does not return to normal on a respiratory function test. Airflow obstruction is caused by the combined action of peripheral airway lesions and emphysematous lesions in various proportions and is progressive. COPD is predicted to be the third leading cause of death worldwide in 2020. Most patients with COPD who require hospitalization due to the disease are thought to be caused by an acute exacerbation of COPD.

  The search for plasma / serum markers and the like, which are indicators of COPD exacerbation, is ongoing (Non-Patent Documents 1 and 2), but no definitive one has been found yet. C-reactive protein (CRP) is also used as an auxiliary marker for COPD exacerbation (Non-Patent Documents 2 and 3), but this is an acute phase protein whose concentration increases rapidly with inflammation, It is a marker of infection / inflammation that triggers exacerbation rather than a marker, and has low disease specificity. A technique for detecting whether or not a COPD patient is aggravated by detecting rhinovirus infection using PCR has also been reported (Patent Document 1), but this method indirectly examines the aggravation from the presence or absence of infection. However, this method can only be applied to a small part of exacerbations caused by rhinovirus. Currently, individual doctors make comprehensive judgments based on indicators such as decreased respiratory function and patient symptoms, and the development of objective and highly accurate indicators for COPD exacerbation is desired.

  A test method for exacerbation sensitivity of COPD patients using prolactin or the like is known (Patent Document 2), but this does not provide an index of exacerbation itself.

US Patent Application No. 2002/0142294 US Patent Application No. 2008/0044843

Barnes P. J., et al., Pulmonary Biomarkers in Chronic Obstructive Pulmonary Disease., (2006) Am. J. Respir. Crit. Care Med., 174: 6-14 Hurst J. R., et al., Use of Plasma Biomarkers at Exacerbation of Chronic Obstructive Pulmonary Disease., (2006) Am. J. Respir. Crit. Care Med., 174: 867-874 Muller B. and Tamm M., Biomarkers in Acute Exacerbation of Chronic Obstructive Pulmonary Disease., (2006) Am. J. Respir. Crit. Care Med., 174: 848-849

  An object of the present invention is to provide a method for detecting an exacerbation index of a COPD patient.

  As a result of intensive studies to solve the above problems, the present inventors have found that increased expression of IL-27 is associated with exacerbation of COPD patients, and have completed the present invention.

That is, the present invention includes the following.
[1] A COPD patient, comprising measuring an expression level of IL-27 protein or a gene encoding the same in a biological sample derived from a COPD patient, and detecting an increase compared to the non-exacerbated expression level of the patient To detect the exacerbation index.
[2] The method according to [1] above, wherein the expression level of IL-27 protein in the biological sample is measured.
[3] The method according to [1] or [2] above, wherein the biological sample is a blood-derived sample.
[4] A kit for detecting a COPD exacerbation index, comprising an antibody that specifically binds to IL-27 protein.

  By using the method of the present invention, an objective index for exacerbation of COPD patients can be obtained.

  Hereinafter, the present invention will be described in detail.

  Siglec-14 is a membrane protein belonging to the immunoglobulin superfamily expressed in cells of the innate immune system (granulocytes, monocytes, etc.) (Angata T., et al., Discovery of Siglec-14, a novel sialic acid receptor undergoing concerted evolution with Siglec-5 in primates., (2006) FASEB J., 20: 1964-1973; Patent 4423394). It is clear that siglec 14 is involved in immune cell activation, and that there are polymorphisms in human siglec 14 gene, and there are humans and non-humans that express siglec 14 protein due to the genetic polymorphism. (Yamanaka M., et al., Deletion polymorphism of SIGLEC14 and its functional implications., (2009) Glycobiology. 19: 841-846; JP 2010-035551 A). When stimulating immune cells using Haemophilus influenza, which is known to cause COPD exacerbation, the present inventors compared immune cells that express Siglec 14 protein with immune cells that do not express this. It has been found that a strong cytokine response is exhibited, and that the genetic polymorphism of the Siglec 14 gene has a strong influence on exacerbation susceptibility in COPD patients (Japanese Patent Application No. 2011-175356). Since Siglec 14 recognizes non-typeable Haemophilus influenzae (NTHi) and activates immune cells, we express Siglec 14 and are activated by NTHi stimulation. We selected genes that are highly expressed in immune cells, and tried to identify genes / proteins that could serve as markers for exacerbation of COPD, and as a result, IL-27 protein, which is a kind of cytokine, and coding for it. Based on this finding, the present invention provides a method for detecting an exacerbation index (exacerbation test) for COPD patients by measuring changes in the expression level of IL-27 as an exacerbation index. provide.

  There is a report that the concentration of IL-27 in sputum and serum is higher in COPD patients than in healthy individuals (Cao J., et al., IL-27 is Elevated in Patients with COPD and Patients with Pulmonary TB and Induces Human Bronchial Epithelial Cells to Produce CXCL10., (2012) Chest., 141: 121-130). However, there have been no reports on changes in IL-27 concentration during exacerbations in COPD patients, and it was first found by the present inventors that IL-27 is a marker for exacerbation of COPD.

  In the method of the present invention, the expression level of IL-27 protein or a gene encoding the same in a biological sample derived from a COPD patient may be measured.

  A patient (COPD patient) diagnosed as a chronic obstructive pulmonary disease (COPD) by a doctor is a suitable test subject in the method of the present invention. Patients with current or past smoking history, cough, sputum, and dyspnea are likely to have COPD. For such patients, a pulmonary function test is performed using the pulmonary function test system CHESTAC (Chest Co., Ltd.), etc., and the 1-second rate (FEV1 / FVC) after bronchodilator administration is 0.7 (70%) The following patients can be diagnosed as patients with chronic obstructive pulmonary disease (COPD). Here, FEV1 is a one-second amount, that is, an exhaled amount that can be exhaled in one second when exhaling as fast as possible. FVC is the vital capacity when forced breathing, that is, when breathing in and breathing out as fast as possible. The COPD patient is a mammal, preferably a human.

  The biological sample used for the measurement in the method of the present invention is collected from a COPD patient who performs an exacerbation test. The biological sample is not limited to, for example, blood (eg, peripheral blood), serum, blood-derived sample such as plasma, oral or nasopharyngeal mucosa, nasal or bronchial aspirate, saliva, breath sample, Sputum etc. are preferred, and blood-derived samples are more preferred.

  The method of the present invention includes, but is not limited to, a biological sample derived from a COPD patient who is in a stable period but whose follow-up is desired, a COPD patient suspected of exacerbation, or a COPD patient diagnosed as exacerbated from symptoms. It is also preferable to carry out as an inspection object. The method of the present invention can be suitably performed, for example, using a biological sample derived from a COPD patient who has passed one week or more from the start of exacerbation as a test target. When testing biological samples derived from COPD patients one week after the start of exacerbation, it is more likely that the expression level of IL-27 has increased during exacerbation, so a more reliable index can be obtained. . Unlike the case of C-reactive protein (CRP), which is thought to reflect bacterial infection, not exacerbation itself, and its expression level decreases within one week from the onset of exacerbation, the expression level of IL-27 is 1 from the onset of exacerbation Maintain an increased state as long as there is an exacerbation even after a week. Therefore, IL-27 is useful as a marker that reflects the presence of exacerbations. “Start of exacerbation” is usually determined as the first date on which exacerbation symptoms appear based on the judgment of a doctor.

  In the present invention, it is necessary to obtain measurement values for a biological sample collected at the time of an exacerbation test for a single patient as well as a biological sample at the time of non-exacerbation. Here, exacerbation of chronic obstructive pulmonary disease (COPD) means that the symptoms of COPD, such as dyspnea, cough, and sputum, suddenly worsen beyond normal physiological fluctuations, and the treatment during non-exacerbation (stable period) The condition that requires change (Japanese Respiratory Society Guidelines, 2009). COPD exacerbations are defined by Anthonisen et al. (Anthonisen NR et al., Ann Intern Med. 1987, 106: 196-204) and Rodriguez-Roisin (Rodriguez-Roisin, R. Chest 2000, 117: 398S-401S). ) Can be determined. In the present invention, the “non-exacerbation” of a COPD patient refers to a time when COPD symptoms such as dyspnea, cough, and sputum in the COPD patient are within the range of daily physiological fluctuations. For COPD patients who may be examined by the method of the present invention, it is preferable to collect a biological sample during non-exacerbation in advance. For example, it is preferable to collect a biological sample at the time of non-exacerbation in advance and store it for future measurement, and measure the biological sample collected at the time of examination and the biological sample at the time of non-exacerbation at the same time. Alternatively, the biological sample at the time of non-exacerbation may be measured in advance, and the measured value may be compared with the measured value of the biological sample at the time of examination.

  In one embodiment of the method of the present invention, the expression level of IL-27 protein in a biological sample at the time of examination of a COPD patient and in a non-exacerbated biological sample can be measured.

  Even if a biological sample is collected from a COPD patient clinically determined to be non-exacerbated according to the above criteria, if the expression level of IL-27 is increased, it is excluded from the non-exacerbated biological sample. It is preferable to do. Such a case may represent an early exacerbation in which no symptoms are shown, an existing exacerbation that has not ended.

  IL-27 protein is a heterodimeric protein in which an IL-27β subunit (also known as EBI3) and an IL-27α subunit (also known as p28 or IL-30) are non-covalently bound. The sequence of the gene and amino acid sequence of human IL-27β subunit is known (GenBank accession number NM — 005755.2) and is shown in SEQ ID NO: 1 (cDNA sequence corresponding to mRNA) and SEQ ID NO: 2, respectively. The gene and amino acid sequence of the human IL-27α subunit is known (GenBank accession number NM — 145659.3) and is shown in SEQ ID NO: 3 (cDNA sequence corresponding to mRNA) and SEQ ID NO: 4, respectively.

  The IL-27β subunit, which is a cytokine constituting the human IL-27 protein to be measured by the method of the present invention, may be composed of the amino acid sequence shown in SEQ ID NO: 2, but is shown in SEQ ID NO: 2. A protein having a deletion, substitution or addition of 1 to 100 (for example, 1 to 50 or 1 to 10) amino acid residues in the amino acid sequence and having cytokine activity, for example, shown in SEQ ID NO: 2 It may be a protein comprising a sequence obtained by removing the signal peptide (N-terminal 1 to 20 residues of SEQ ID NO: 2) from the amino acid sequence. The IL-27α subunit, which is a cytokine constituting the human IL-27 protein to be measured in the method of the present invention, may be composed of the amino acid sequence shown in SEQ ID NO: 4 or shown in SEQ ID NO: 4. A protein having cytokine activity, such as shown in SEQ ID NO: 4, comprising a sequence comprising deletion, substitution or addition of 1 to 100 (for example, 1 to 50 or 1 to 10) amino acid residues in the amino acid sequence Or a protein comprising a sequence obtained by removing a signal peptide (N-terminal residues 1 to 28 of SEQ ID NO: 4) from the amino acid sequence.

The expression level of IL-27 protein can be measured by quantifying IL-27 protein in a biological sample using a known protein measurement method based on ELISA, flow cytometry, or the like. For example, IL-27 protein in a biological sample is measured based on ELISA using an antibody that specifically binds to IL-27 protein, IL-27β subunit, or IL-27α subunit. can do. The antibody that specifically binds to IL-27 protein, IL-27β subunit, or IL-27α subunit used for the measurement in the present invention may be a monoclonal antibody or a polyclonal antibody. For measurement, various commercially available anti-IL-27 antibodies and human or mouse IL-27 ELISA assay kits can also be used. For example, from R & D Systems, a polyclonal antibody prepared using a recombinant fusion protein in which an IL-27α subunit and an IL-27β subunit are linked by a linker peptide as an antigen, and an ELISA for detecting IL-27 using the same. Kits can be purchased. An antibody that specifically binds to IL-27 protein, IL-27β subunit, or IL-27α subunit uses IL-27 protein, IL-27β subunit, IL-27α subunit, or a fragment thereof as an antigen. It can also be prepared by known immunological techniques or genetic engineering techniques. For example, polyclonal antibodies against IL-27 protein, IL-27β subunit, or IL-27α subunit immunize animals such as mice, rats, rabbits, goats, sheep, donkeys using them as antigens, It can be obtained by collecting serum after a certain period of time and purifying it with a column or the like on which protein A, protein G, antigen protein, antigen peptide, etc. are immobilized. Further, a monoclonal antibody against IL-27 protein, IL-27β subunit, or IL-27α subunit is immunized in the same manner as the above polyclonal antibody, and after a certain period of time, for example, spleen cells are collected and Cell fusion with myeloma cells, screening of the obtained hybridoma, selecting a hybridoma producing an antibody exhibiting specific binding to the antigen protein used, and preparing from the culture or culture supernatant be able to. In addition, antigen-binding fragments (Fab, F (ab ′) _2, etc.) of these antibodies can be used for measurement in the same manner as for all antibodies. The quantification of IL-27 protein in a biological sample can also be performed by a multiplex bead array method (Procarta Cytokine Assay Kit, Affymetrix) using a cytokine assay kit.

  The measured value of IL-27 protein in the biological sample thus obtained can be used as the expression level of IL-27 protein. The expression level of IL-27 protein may be expressed as a measured value such as a concentration, or may be expressed as a relative value.

  The expression level of IL-27 protein in a biological sample at the time of examination of a COPD patient obtained as described above was compared with the expression level of IL-27 protein measured in the same manner for a biological sample at the time of non-exacerbation of the same patient. Compare.

  In another embodiment of the method of the present invention, the expression level of a gene encoding IL-27 protein in a biological sample at the time of exacerbation test for COPD patients and a biological sample at the time of non-exacerbation may be measured. As used herein, “gene encoding IL-27 protein” means a gene encoding IL-27β subunit or IL-27α subunit constituting IL-27 protein. The gene encoding the IL-27β subunit to be measured may be composed of the base sequence shown in SEQ ID NO: 1, or 1 to 10 (for example, 1 to 1) in the base sequence shown in SEQ ID NO: 1. It may be a sequence comprising a deletion, substitution or addition of 5) bases, and may encode a protein having cytokine activity. The gene encoding the IL-27α subunit may be a nucleic acid having the base sequence shown in SEQ ID NO: 3, or 1 to 10 (eg, 1 to 5) in the base sequence shown in SEQ ID NO: 3. Or a nucleic acid encoding a protein having cytokine activity. The gene encoding the IL-27α subunit may also be a nucleic acid that hybridizes with the nucleotide sequence shown in SEQ ID NO: 3 under stringent conditions and encodes a protein having cytokine activity. A protein comprising a nucleotide sequence having the identity of 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more with respect to the base sequence shown in 3, and having cytokine activity It may be a nucleic acid. In the present invention, “stringent conditions” refers to conditions under which a so-called specific hybrid is formed. For example, highly homologous nucleic acids, that is, DNA having 90% or more, preferably 95% or more homology. Examples include conditions in which nucleic acids hybridize with each other and nucleic acids with lower homology do not hybridize with each other. More specifically, the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM, the temperature is 25 to 70 ° C., preferably 50 to 70 ° C., more preferably 55 to 65 ° C., This refers to conditions at a formamide concentration of 0-50%, preferably 20-50%, more preferably 35-45%. Furthermore, under stringent conditions, the washing conditions of the filter after hybridization are usually 15 to 600 mM, preferably 50 to 600 mM, more preferably 300 to 600 mM, and a temperature of 50 to 70 ° C., preferably It is 55-70 degreeC, More preferably, it is 60-65 degreeC. The “nucleic acid” may be DNA or RNA, and may contain a modified base in part.

  The expression level of a gene encoding IL-27 protein can be measured by measuring the amount of mRNA expressed from the gene of a nucleic acid sample extracted from a biological sample, preferably total RNA. Total RNA can be extracted by a conventional method using a commercially available RNA extraction kit or the like. Measurement of the amount of mRNA derived from a gene encoding IL-27β subunit or IL-27α subunit can specifically amplify mRNA transcribed from the gene encoding IL-27β subunit or IL-27α subunit It can be performed by quantitative PCR using PCR primer pairs. Alternatively, such mRNA amount can be measured based on Northern blotting using a probe specific for a gene encoding IL-27β subunit or IL-27α subunit. A person skilled in the art appropriately designs an appropriate primer or probe based on the nucleotide sequence of a gene encoding IL-27β subunit or IL-27α subunit, for example, the nucleotide sequence shown in SEQ ID NO: 1 or 3. be able to.

  The amount of mRNA expressed from the gene encoding IL-27β subunit or IL-27α subunit in the biological sample measured as described above is used as the expression level of the gene encoding IL-27 protein. Can do. The expression level may be expressed as a measured value such as a concentration, or may be expressed as a relative value.

  Next, the expression level of the gene encoding IL-27 protein in the biological sample at the time of exacerbation test of COPD patient is compared with the expression level of the gene encoding IL-27 protein in the biological sample at the time of non-exacerbation of the same patient. .

  In the method of the present invention, the expression level of IL-27 protein or a gene encoding IL-27 protein in a biological sample at the time of exacerbation test of a COPD patient is expressed as the corresponding expression level in the biological sample at the time of non-exacerbation of the same patient. It is determined whether the expression level in the biological sample at the time of the exacerbation test is higher than the expression level in the biological sample at the time of the non-exacerbation. As a result, if the expression level in the biological sample at the time of the test is higher than the expression level in the non-exacerbated biological sample, it is highly likely that the COPD patient is exacerbated in COPD. Furthermore, when the expression level in the biological sample at the time of the test is increased by 5% or more, preferably 10% or more, more preferably 20% or more compared to the expression level in the biological sample at the time of non-exacerbation, COPD exacerbation It can be said that the possibility of occurrence is particularly high.

  As described above, by measuring the expression level of IL-27 protein or a gene encoding the same in a biological sample derived from a COPD patient, and detecting an increase compared to the expression level in the non-exacerbation period of the patient, The detection result can be used as an objective index indicating the possibility of COPD patient exacerbation. The present invention provides a method for detecting an exacerbation index of such a COPD patient.

  According to the method of the present invention, for example, an objective index indicating aggravation can be detected in a patient who cannot be determined to be COPD based on symptoms, thereby supporting the diagnosis of COPD. Furthermore, if the method of the present invention is used, the exacerbation index according to the present invention can be obtained as an index that can be used to determine the effectiveness of the exacerbation suppression / amelioration treatment for COPD patients being treated. In the method of the present invention, an increase in IL-27 can be a more reliable indicator of exacerbation, particularly when examining COPD patients from one week after the exacerbation start date. Therefore, the method of the present invention is particularly advantageous when monitoring the progress of COPD exacerbation over a long period of time (preferably for a week or more) for purposes such as determining the effectiveness of treatment.

  The present invention also provides a kit for detecting a COPD exacerbation index, comprising an antibody that specifically binds to IL-27 protein. The antibody that specifically binds to IL-27 protein may be a monoclonal antibody or a polyclonal antibody, and is the same as that described above for measuring IL-27 protein. In addition to antibodies that specifically bind to IL-27 protein, the kit includes other reagents, for example, ELISA assay reagents such as secondary antibodies for antibody detection, buffers, solid phases (for example, beads), COPD It may further include an instruction manual for detecting an exacerbation index.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Selection of serum marker candidates for exacerbation (1) Preparation of NTHi dead bacteria, stimulation of THP-1 cells and preparation of RNA Non-typeable Haemophilus influenzae (NTHi) 2019 strain And cultured in a minimal medium supplemented with 0.5 mM N-acetylneuraminic acid as described in the literature (Greiner LL et al., Infect Immun. 2004, 72: 4249-60; Taylor RE, et al., J Exp Med. 2010, 207: 1637-46), and then sterilized by heating at 60 ° C. for 30 minutes.

  A strain obtained by stably introducing the siglec 14 gene (SEQ ID NO: 5) cDNA and the siglec 5 gene (SEQ ID NO: 6) cDNA into human monocytic leukemia cell THP-1 Each strain was prepared (Yamanaka M., et al., (2009) Glycobiology. 19: 841-846). Cells into which the Siglec 14 gene has been introduced and expresses are models of human monocytes that are homozygous for the Siglec 14 wild type allele. Cells in which the Siglec 5 gene has been introduced and expressed are models of human monocytes that are homozygous for the Siglec 14 null allele.

Cells of these strains were suspended at 1 × 10 ⁶ cells / ml in macrophage / monocyte serum-free medium Macrophage-SFM (Invitrogen), and 2.5 ml was dispensed into each well of a 6-well plate (2.5 × 10 ⁵ Cells / well). To these cells, the NTHi culture heat-sterilized as described above is added (2.5 × 10 ⁸ NTHi / well; the control is prepared without the addition of NTHi) and 6% in a carbon dioxide incubator (37 ° C., 5% CO ₂ ). Incubate for hours. After culturing, cells were collected by centrifugation, and total RNA was extracted using RNeasy Mini (Qiagen).

(2) Gene expression analysis using DNA microarray
Gene expression analysis (Affymetrix) in each cell using a DNA microarray was performed according to the following procedure.

i) Quantification of RNA and confirmation of quality The total RNA extracted from the cells in (1) above was analyzed using 2100 Bioanalyzer (Agilent) to confirm the quality of RNA (less degradation). The concentration and purity were measured with a spectrophotometer NanoDrop.

ii) Preparation and fragmentation of biotin-labeled RNA Using the above-mentioned total RNA 250 ng as a template, GeneChip (registered trademark) 3 'IVT Express Kit (Affymetrix) was used to sequentially synthesize first- and second-strand cDNA. Furthermore, biotin-labeled RNA was prepared by in vitro transcription and fragmented according to the protocol.

iii) Hybridization to DNA microarray The fragmented biotin-labeled RNA was hybridized (45 ° C, 16 hours) to DNA microarray Human Genome U133 Plus 2.0 (Affymetrix). For hybridization, a hybridization oven GeneChip Hybridization Oven 640 (Affymetrix) was used.

iv) Detection of hybridized biotin-labeled RNA Using GeneChip Hybridization, Wash, and Stain Kit (Affymetrix) as a reagent and GeneChip Fluidics Station 450 (Affymetrix) as a device, the hybridized fragmented biotin-labeled RNA is fluorescent-labeled streptogram. Avidin was bound, washed, and images were scanned using the scanner GeneChip Scanner 3000 7G (Affymetrix). The software GeneChip Operating Software (Affymetrix) was used to control the GeneChip Fluidics Station 450 and GeneChip Scanner 3000 7G.

v) Quantification of gene expression
Using GeneChip Operating Software, the fluorescence intensity of each spot was quantified from the scanned image, and the quantified value was standardized.

(3) Selection of marker candidates based on gene expression patterns The samples examined for gene expression as described above are as follows.
Sample A: Siglec 14 gene transfer / THP-1 cells, NTHi (+)
Sample B: Siglec 14 gene transfer / THP-1 cells, NTHi (-)
Sample C: Siglec 5 gene transfer / THP-1 cells, NTHi (+)
Sample D: Siglec 5 gene transfer / THP-1 cells, NTHi (-)
Here, comparing the gene expression patterns of sample A and sample B, A shows more than twice the expression of B, and comparing the gene expression patterns of sample A and sample C, A Genes that expressed more than about 2 times C were extracted. In addition, the gene expression level is above a certain level, the protein that is the gene product is secreted, the protein is not synthesized in the liver, and the protein measurement method is established. Multiple cleared genes were selected and designated as “COPD exacerbation serum marker candidates”. The selected “serum marker candidate for COPD exacerbation” gene group included the gene EBI3 (also referred to as IL-27β) encoding the β subunit of the heterodimeric protein IL-27.

[Example 2] Quantitative analysis of marker candidates using patient-derived exacerbated / non-exacerbated paired serum samples (1) Recruitment of patients and preparation of serum Among the outpatients who visited Nippon Medical University Respiratory Care Clinic, Those who meet the conditions were asked whether they were willing to participate in the study: (a) current or past smoking history, and (b) cough, sputum, and dyspnea. Informed consent was obtained from patients who indicated their intention to participate in the study. Blood was collected from patients who obtained informed consent at the time of stable (non-exacerbation) and at the time of exacerbation, and serum was prepared according to a conventional method. Pulmonary function tests are performed using the pulmonary function test system CHESTAC (Chest Co., Ltd.), and patients with a 1-second rate (FEV1 / FVC) of 0.7 (70%) or less after administration of bronchodilator according to conventional methods are chronic obstructive pulmonary Diagnosed as a patient with COPD.

  In addition, information to that effect was registered in serum samples collected from one week after the start of exacerbation. The date of exacerbation was determined by the physician based on the symptoms that appeared in the patient.

(2) Quantification of marker candidates Regarding the genes selected as “serum marker candidates for COPD exacerbation” in Example 1, using the multiplex bead array method (Procarta Cytokine Assay Kit, Human By Request; Panomics / Affymetrix), the following The protein corresponding to each expression product was measured by the procedure (n = 39). This method is a method of simultaneously quantifying multiple measurement objects in the same sample by applying Luminex's xMAP technology. Measurement was carried out according to the protocol of Panomics / Affymetrix (Procarta Cytokine Assay Kit, User Manual Specifically for Serum and Plasma Samples; P / N 10483).

i) Preparation of antibody beads Add 50 μl of each antibody bead mixture (each labeled with a specific amount of fluorescent dye) to each multi-screen vacuum manifold (Millipore) with a filter plate. Then, the liquid was removed by aspiration, washed with Wash Buffer, and aspirated to remove the liquid.

ii) Adsorption to antibody beads to be analyzed Add 25 μl of Assay Buffer to each well, and then add 25 μl of serum or standard mixture for quantification (Create a 4-fold dilution series using Standard Buffer and Human Serum with Human Assay Buffer) And incubated at room temperature for 60 minutes with shaking. The liquid was removed by aspiration, washed with Wash Buffer, and aspirated to remove the liquid.

iii) Binding of biotinylated antibody 25 μl of biotinylated detection antibody mixture was added to each well and incubated at room temperature for 30 minutes with shaking. The liquid was removed by aspiration, washed with Wash Buffer, and aspirated to remove the liquid.

iv) Binding with streptavidin-fluorescent dye conjugate 50 μl of streptavidin-R-phycoerythrin solution was added to each well and incubated at room temperature for 30 minutes with shaking. The liquid was removed by aspiration, washed with Wash Buffer, and aspirated to remove the liquid.

v) Measurement and quantification of fluorescence intensity 120 μl of Reading Buffer was added to each well, shaken at room temperature for 5 minutes, and subjected to fluorescence measurement of each bead. Quantify the analyte concentration in each sample using the Bio-Plex workstation (BIO-RAD) to measure fluorescence intensity and Bio-Plex Manager software 6.0 (BIO-RAD) to analyze the data. did.

(3) Statistical analysis Measured values of each measurement target in paired sera during exacerbation and non-exacerbation were compared. Since the distribution of measured values does not follow the normal distribution, the Wilcoxon sign rank test was adopted as a nonparametric test method. The significance level was set to 0.05.

  As a result, it was shown that the serum protein concentration of IL-27 protein increased significantly during exacerbation (p value: 0.0472 (<0.05), one-sided test). In the patient population measured here, IL-27 at the time of exacerbation showed a maximum increase of about 148% per patient compared to non-exacerbation (non-exacerbation: 397.25, exacerbation: 988.19) It was. In addition, the proportion of patients whose IL-27 measurement at the time of exacerbation showed an increase of 10% or more compared to non-exacerbation was over 45% of the COPD exacerbated patients who measured.

  Furthermore, C-reactive protein (CRP), an existing CDPD exacerbation assisting marker, was compared with IL-27. The measurement of high sensitivity CRP was outsourced to BML Co., Ltd. N-latex CRP II from Siemens Healthcare Diagnostics Co., Ltd. was used for CRP quantification.

  Both CRP and IL-27 were significantly higher in the case of exacerbations in all counts (CRP: 37 cases, IL-27: 39 cases) (one-sided test, p <0.05). On the other hand, when limited to serum samples collected one week after the onset of exacerbation, IL-27 (25 cases) was higher than in non-exacerbated cases (significant for both one-sided and two-sided tests; p-value: 0.0027 (one-sided), 0.0053 (two-sided) (<0.01)) and CRP (24 cases) were not high (one-sided and two-sided tests were not significant; p> 0.05).

  In addition, cases with purulent sputum (suspected bacterial infection, 8 cases) were significantly higher in CRP than in other cases, but IL-27 was significantly higher. Not shown. This indicates that the increase in exacerbation of IL-27 is not limited to the time of bacterial infection.

  These results indicate that IL-27 is a marker that reflects the presence of COPD exacerbations. For this reason, IL-27 was thought to be useful as an index for evaluating drug efficacy during treatment.

  The method of the present invention enables an objective diagnosis of exacerbation of COPD patients, and is expected to lead to improvement in patient QOL and prognosis, and effective use of medical resources.

Claims (3)

An exacerbation of a COPD patient, comprising measuring the expression level of IL-27 protein or a gene encoding the same in a biological sample derived from a COPD patient, and detecting an increase compared to the expression level of the patient during non-exacerbation A method for detecting an index, wherein the biological sample is a blood-derived sample . The method according to claim 1, wherein the expression level of IL-27 protein in the blood-derived sample is measured.   A kit for detecting a COPD exacerbation index, comprising an antibody that specifically binds to IL-27 protein.