JPWO2007145274A1 - Lung injury test method and test kit by measuring cytochrome c - Google Patents

Lung injury test method and test kit by measuring cytochrome c Download PDF

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JPWO2007145274A1
JPWO2007145274A1 JP2008521247A JP2008521247A JPWO2007145274A1 JP WO2007145274 A1 JPWO2007145274 A1 JP WO2007145274A1 JP 2008521247 A JP2008521247 A JP 2008521247A JP 2008521247 A JP2008521247 A JP 2008521247A JP WO2007145274 A1 JPWO2007145274 A1 JP WO2007145274A1
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cytochrome
lung injury
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disease
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JP5322638B2 (en
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和善 桑野
和善 桑野
宗夫 青山
宗夫 青山
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Eisai R&D Management Co Ltd
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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

Abstract

間質性肺疾患などにおける肺傷害の検査方法、及び、肺傷害を伴う肺疾患の検査方法を提供する。気管支肺胞洗浄液中のチトクロムｃ濃度を測定することにより間質性肺疾患などにおける肺傷害の検査が可能となる。また、肺傷害を伴う肺疾患を検査できる。A method for examining lung injury in interstitial lung disease and the like, and a method for examining lung disease accompanied by lung injury are provided. By measuring the cytochrome c concentration in the bronchoalveolar lavage fluid, it becomes possible to examine lung injury in interstitial lung disease and the like. In addition, lung diseases associated with lung injury can be examined.

Description

本発明は、肺疾患における肺傷害の検査方法および検査キットに関する。さらに詳しくは、間質性肺疾患または急性呼吸窮迫症候群等の肺疾患における肺傷害の検査方法および検査キットに関する。また、肺傷害を伴う肺疾患の検査方法及び検査キットに関する。   The present invention relates to a test method and test kit for lung injury in lung diseases. More particularly, the present invention relates to a method and a test kit for lung injury in lung diseases such as interstitial lung disease or acute respiratory distress syndrome. Moreover, it is related with the test | inspection method and test kit of the lung disease which accompanies a lung injury.

肺疾患には、肺癌、縦隔腫瘍、呼吸器感染症（肺炎，肺抗酸菌症，肺真菌症）、慢性閉塞性肺疾患、間質性肺疾患（間質性肺炎）、急性呼吸窮迫（促迫）症候群（ＡＲＤＳ）、気管支喘息・アレルギー性肺疾患、自然気胸、サルコイドーシス、気管支拡張症、肺血栓塞栓症、睡眠時無呼吸症候群（外来簡易検査）等があり、さまざまな診断方法がある。特に、間質性肺疾患は、肺胞の壁（間質）に炎症がおきる疾患を総称するもので、その中でも繊維化を起こしやすい疾患を特に間質性肺炎と呼んでいる。間質性肺炎は、肺胞の形も不規則になって、肺全体が少し固くなっていく。その結果、肺のふくらみが悪くなり肺活量がおちると同時に、酸素の吸収効率も悪くなってゆき、息苦しくなったり、咳が出たりして、さらに進行すると、 肺は線維性成分の固まりとなり、この部分での肺としての機能が失われる。この間質性肺炎には、特発性肺線維症、非特異性間質性肺炎、急性間質性肺炎、特発性器質化肺炎、剥離性間質性肺炎、呼吸細気管支炎関連性間質性肺疾患、リンパ球性間質性肺炎等の疾患があるが、肺胞細胞の傷害の程度により、治療方法を変えることが疾患の早期治癒に重要である。   Lung diseases include lung cancer, mediastinal tumor, respiratory infection (pneumonia, pulmonary mycobacterial disease, pulmonary mycosis), chronic obstructive pulmonary disease, interstitial lung disease (interstitial pneumonia), acute respiratory distress There are various diagnosis methods such as (urge) syndrome (ARDS), bronchial asthma / allergic lung disease, spontaneous pneumothorax, sarcoidosis, bronchiectasis, pulmonary thromboembolism, sleep apnea syndrome (outpatient simple examination) . In particular, interstitial lung disease is a general term for diseases in which inflammation occurs in the alveolar wall (interstitium). Among them, a disease that easily causes fibrosis is particularly called interstitial pneumonia. In interstitial pneumonia, the shape of the alveoli becomes irregular and the whole lungs become a little hard. As a result, the lungs swell and the vital capacity falls, and at the same time, the oxygen absorption efficiency also deteriorates.They become stuffy or cough, and if they progress further, the lungs become a mass of fibrous components. The function as a lung in the part is lost. This interstitial pneumonia includes idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, acute interstitial pneumonia, idiopathic organized pneumonia, exfoliative interstitial pneumonia, respiratory bronchiolitis-related interstitial lung Although there are diseases such as diseases and lymphocytic interstitial pneumonia, changing the treatment method depending on the degree of alveolar cell injury is important for the early cure of the disease.

急性呼吸窮迫症候群は、ショック肺とも呼ばれ、さまざまな原因によって引き起こされる急性の呼吸困難、重症の低酸素血症、肺損傷の総称をいう。肺の間質や肺胞腔に水分と細胞浸潤がみられ、酸素化（血液中に酸素を取り込むこと）が損なわれる症状が見られる。その本体は、血管内皮細胞が損なわれて血管の透過性が進み、その結果もたらされる急性肺水腫と考えられている。   Acute respiratory distress syndrome, also called shocked lung, is a general term for acute dyspnea, severe hypoxemia, and lung injury caused by various causes. Water and cell infiltration are seen in the lung interstitium and alveolar space, and oxygenation (incorporation of oxygen into the blood) is impaired. The body is considered acute pulmonary edema resulting from impaired vascular endothelial cells and increased vascular permeability.

ところが、現在、肺傷害（肺胞細胞の傷害）の程度を容易に検査する方法がなく、特に間質性肺疾患や急性呼吸窮迫症候群において、肺傷害を表すマーカーが望まれている。   However, there is currently no method for easily examining the degree of lung injury (alveolar cell injury), and a marker indicating lung injury is particularly desired in interstitial lung disease and acute respiratory distress syndrome.

一方、チトクロムｃは、ミトコンドリアにおける電子伝達系の重要な蛋白質として知られているが、細胞がアポトーシスの引き金となる刺激に曝され、アポトーシスの状態になると、ミトコンドリアにあったチトクロムｃが急速にサイトゾルへと放出されることが報告されている（非特許文献１）。そしてサイトゾルのチトクロムｃは、アポトーシスのキーファクターであるカスパーゼ(caspase)-3の活性化に関与し、チトクロムｃの増加が、アポトーシス進行と関係することが報告されている（非特許文献２）。   On the other hand, cytochrome c is known as an important protein of the electron transport system in mitochondria, but when cells are exposed to a stimulus that triggers apoptosis and becomes apoptotic, cytochrome c in mitochondria rapidly becomes a site. It has been reported that it is released into a sol (Non-Patent Document 1). Cytosolic cytochrome c is involved in the activation of caspase-3, which is a key factor of apoptosis, and it has been reported that an increase in cytochrome c is associated with the progression of apoptosis (Non-patent Document 2). .

チトクロムｃを測定することにより種々の診断を行うことが知られている（特許文献１−２）。
国際公開０１／３５０９３号パンフレット 特開２００３−０２８８６０号 Dinsdale D. et al., American J. Pathol. 155:607-18, 1999 Medina V. et al., Cancer Research 57:3697-707, 1999 It is known to perform various diagnoses by measuring cytochrome c (Patent Documents 1-2).
International publication 01/35093 pamphlet JP 2003-028860 A Dinsdale D. et al., American J. Pathol. 155: 607-18, 1999 Medina V. et al., Cancer Research 57: 3697-707, 1999

本発明の課題は、肺傷害（肺胞細胞の傷害）の検査方法および検査キットを提供することである。   An object of the present invention is to provide a test method and test kit for lung injury (alveolar cell injury).

また、他の課題は、肺傷害を伴う肺疾患、特に間質性肺疾患や急性呼吸窮迫症候群の検査方法および検査キットを提供することである。   Another object is to provide a test method and test kit for lung diseases associated with lung injury, particularly interstitial lung disease and acute respiratory distress syndrome.

本発明者らは、肺胞細胞の傷害にアポトーシスが関与していると予測し、研究を行った結果、気管支肺洗浄液中の、アポトーシス関連タンパクであるチトクロムｃを測定することにより肺胞細胞の傷害を検査できることを見出し、発明を完成するに至った。   The present inventors have predicted that apoptosis is involved in the injury of alveolar cells, and as a result of conducting research, the inventors measured cytochrome c, an apoptosis-related protein in bronchopulmonary lavage fluid, to determine the alveolar cell's damage. The inventor found that the injury could be inspected, and completed the invention.

すなわち本発明は、以下に関する。
［１］ 肺疾患における肺傷害の検査方法であって、採取した体液を含む溶液中のチトクロムｃを定量することにより肺傷害を検査することを特徴とする方法。
［２］ 肺傷害が間質性肺疾患における肺傷害である、項１記載の方法。
［３］ 肺傷害が急性呼吸窮迫症候群における肺傷害である、項１記載の方法。
［４］ 定量が免疫化学的方法による項１〜３のいずれか１項に記載の方法。
［５］ 以下の工程を含む項４記載の方法。
（１）採取した体液中のチトクロムｃを免疫化学的方法により定量する工程
（２）定量した結果、正常値と比較して高値のときに肺傷害と判定する工程
［６］ 免疫化学的方法が抗チトクロムｃ抗体とチトクロムｃの反応による方法である、項４又は５に記載の方法。
［７］ 抗チトクロムｃ抗体とチトクロムｃの反応を酸性領域の緩衝液中で行う、項６記載の方法。
［８］ 酸性領域がｐＨ３．０からｐＨ６．０である、項７記載の方法。
［９］ 体液を含む溶液が気管支肺胞洗浄液である項１〜８のいずれか１項に記載の方法。That is, the present invention relates to the following.
[1] A method for examining lung injury in a lung disease, wherein the lung injury is examined by quantifying cytochrome c in a solution containing a collected body fluid.
[2] The method according to Item 1, wherein the lung injury is lung injury in interstitial lung disease.
[3] The method according to Item 1, wherein the lung injury is lung injury in acute respiratory distress syndrome.
[4] The method according to any one of items 1 to 3, wherein the quantification is performed by an immunochemical method.
[5] The method according to item 4, comprising the following steps.
(1) A step of quantifying cytochrome c in the collected body fluid by an immunochemical method (2) A step of determining a lung injury when the value is higher than a normal value as a result of the quantification [6] Item 6. The method according to Item 4 or 5, which is a method based on a reaction between an anti-cytochrome c antibody and cytochrome c.
[7] The method according to item 6, wherein the reaction between the anti-cytochrome c antibody and cytochrome c is performed in an acidic buffer solution.
[8] The method according to Item 7, wherein the acidic region is pH 3.0 to pH 6.0.
[9] The method according to any one of Items 1 to 8, wherein the solution containing the body fluid is bronchoalveolar lavage fluid.

［１０］ 肺傷害検査用キットであって、採取した体液を含む溶液中のチトクロムcを定量するための試薬を含むことを特徴とするキット。
［１１］ 肺傷害が間質性肺疾患における肺傷害である、項１０記載のキット。
［１２］ 肺傷害が急性呼吸窮迫症候群における肺傷害である、項１０記載のキット。
［１３］ 定量が免疫化学的方法による項１０〜１２のいずれか１項に記載のキット。
［１４］ 免疫化学的方法が抗チトクロムｃ抗体とチトクロムｃの反応による方法である、項１３に記載のキット。
［１５］ 抗チトクロムｃ抗体とチトクロムｃの反応を酸性領域の緩衝液中で行うための緩衝液を含む、項１４記載のキット。
［１６］ 酸性領域がｐＨ３．０からｐＨ６．０である、項１５記載のキット。
［１７］ 体液を含む溶液が気管支肺胞洗浄液である項１０〜１６のいずれか１項に記載のキット。[10] A kit for lung injury testing, comprising a reagent for quantifying cytochrome c in a solution containing a collected body fluid.
[11] The kit according to Item 10, wherein the lung injury is lung injury in interstitial lung disease.
[12] The kit according to Item 10, wherein the lung injury is lung injury in acute respiratory distress syndrome.
[13] The kit according to any one of Items 10 to 12, wherein the quantification is performed by an immunochemical method.
[14] The kit according to item 13, wherein the immunochemical method is a method based on a reaction between an anti-cytochrome c antibody and cytochrome c.
[15] The kit according to Item 14, comprising a buffer for performing the reaction between the anti-cytochrome c antibody and cytochrome c in a buffer in an acidic region.
[16] The kit according to Item 15, wherein the acidic region is pH 3.0 to pH 6.0.
[17] The kit according to any one of Items 10 to 16, wherein the solution containing the body fluid is bronchoalveolar lavage fluid.

［１８］ 肺傷害を伴う肺疾患を検査する方法であって、採取した体液を含む溶液中のチトクロムｃを定量することにより肺傷害を伴う肺疾患を検査することを特徴とする方法。
［１９］ 肺疾患が間質性肺疾患である、項１８記載の方法。
［２０］ 肺疾患が急性呼吸窮迫症候群である、項１８記載の方法。
［２１］ 定量が免疫化学的方法による項１８〜２０のいずれか１項に記載の方法。
［２２］ 以下の工程を含む項２１に記載の方法。
（１）採取した体液中のチトクロムｃを免疫化学的方法により定量する工程
（２）定量した結果、正常値と比較して高値のときに肺傷害を伴う肺疾患と判定する工程
［２３］ 免疫化学的方法が抗チトクロムｃ抗体とチトクロムｃの反応による方法である、項２１又は２２記載の方法。
［２４］ 抗チトクロムｃ抗体とチトクロムｃの反応を酸性領域の緩衝液中で行う、項２３記載の方法。
［２５］ 酸性領域がｐＨ３．０からｐＨ６．０である、項２４記載の方法。
［２６］ 体液を含む溶液が気管支肺胞洗浄液である項１８〜２５のいずれか１項に記載の方法。[18] A method for examining a pulmonary disease associated with lung injury by quantifying cytochrome c in a solution containing a collected body fluid, wherein the pulmonary disease associated with lung injury is examined.
[19] The method according to item 18, wherein the lung disease is interstitial lung disease.
[20] The method according to Item 18, wherein the lung disease is acute respiratory distress syndrome.
[21] The method according to any one of items 18 to 20, wherein the quantification is performed by an immunochemical method.
[22] The method of claim | item 21 including the following processes.
(1) A step of quantifying cytochrome c in the collected body fluid by an immunochemical method (2) A step of determining a lung disease accompanied by lung injury when the quantification results in a higher value compared to the normal value [23] Item 23. The method according to Item 21 or 22, wherein the chemical method is a method of reacting an anti-cytochrome c antibody with cytochrome c.
[24] The method according to item 23, wherein the reaction between the anti-cytochrome c antibody and cytochrome c is carried out in an acidic buffer solution.
[25] The method according to item 24, wherein the acidic region is pH 3.0 to pH 6.0.
[26] The method according to any one of items 18 to 25, wherein the solution containing body fluid is bronchoalveolar lavage fluid.

［２７］ 肺傷害を伴う肺疾患の検査用キットであって、採取した気管支肺胞洗浄液中のチトクロムcを定量するための試薬を含むことを特徴とするキット。
［２８］ 肺疾患が間質性肺疾患である、項２７記載のキット。
［２９］ 肺疾患が急性呼吸窮迫症候群である、項２７記載のキット。
［３０］ 定量が免疫化学的方法による項２７〜２９のいずれか１項に記載のキット。
［３１］ 免疫化学的方法が抗チトクロムｃ抗体とチトクロムｃの反応による方法である、項３０記載のキット。
［３２］ 抗チトクロムｃ抗体とチトクロムｃの反応を酸性領域の緩衝液中で行うための緩衝液を含む、項３１記載のキット。
［３３］ 酸性領域がｐＨ３．０からｐＨ６．０である、項３２記載のキット。
［３４］ 体液を含む溶液が気管支肺胞洗浄液である項２７〜３３のいずれか１項に記載のキット。[27] A kit for examining a lung disease accompanied by lung injury, comprising a reagent for quantifying cytochrome c in a collected bronchoalveolar lavage fluid.
[28] The kit according to item 27, wherein the lung disease is interstitial lung disease.
[29] The kit according to item 27, wherein the pulmonary disease is acute respiratory distress syndrome.
[30] The kit according to any one of items 27 to 29, wherein the quantification is performed by an immunochemical method.
[31] The kit according to item 30, wherein the immunochemical method is a method based on a reaction between an anti-cytochrome c antibody and cytochrome c.
[32] The kit according to item 31, comprising a buffer solution for performing the reaction between the anti-cytochrome c antibody and cytochrome c in an acidic region buffer solution.
[33] The kit according to Item 32, wherein the acidic region is pH 3.0 to pH 6.0.
[34] The kit according to any one of items 27 to 33, wherein the solution containing body fluid is bronchoalveolar lavage fluid.

肺疾患患者の気管支肺胞洗浄液に含まれるチトクロムｃの定量結果を示す（マンホイットニー順位和検定(Mann-Whitney U-test)による疾患群間における有意差も合わせて示す）。ARDS：急性呼吸窮迫（促迫）症候群、IPF：特発性肺線維症、NSIP：非特異的間質性肺炎、COP：持続性器質化肺炎、CVD-IP：膠原病由来の間質性肺炎、HP：過敏性肺炎、SA：サルコイドーシスThe quantitative result of the cytochrome c contained in the bronchoalveolar lavage fluid of a lung disease patient is shown (The significant difference between the disease groups by Mann-Whitney U-test is also shown collectively.). ARDS: acute respiratory distress syndrome, IPF: idiopathic pulmonary fibrosis, NSIP: nonspecific interstitial pneumonia, COP: persistent organizing pneumonia, CVD-IP: interstitial pneumonia derived from collagen disease, HP : Hypersensitivity pneumonia, SA: Sarcoidosis 肺疾患患者の気管支肺胞洗浄液に含まれるチトクロムｃの定量結果を示す（分散分析（one way ＡＮＯＶＡ）および多重比較（post hoc analysis）による疾患群間における有意差もあわせて示す）。ARDS：急性呼吸窮迫（促迫）症候群、IPF：特発性肺線維症、NSIP：非特異的間質性肺炎、COP：持続性器質化肺炎、CVD-IP：膠原病由来の間質性肺炎、HP：過敏性肺炎、SA：サルコイドーシスThe quantitative result of the cytochrome c contained in the bronchoalveolar lavage fluid of a lung disease patient is shown (it also shows the significant difference between the disease groups by a variance analysis (one way ANOVA) and multiple comparison (post hoc analysis)). ARDS: acute respiratory distress syndrome, IPF: idiopathic pulmonary fibrosis, NSIP: nonspecific interstitial pneumonia, COP: persistent organizing pneumonia, CVD-IP: interstitial pneumonia derived from collagen disease, HP : Hypersensitivity pneumonia, SA: Sarcoidosis 図２の結果を、ARDS群以外の間質性肺炎の各疾患の差を示したものである。The result of FIG. 2 shows the difference of each disease of interstitial pneumonia other than the ARDS group. モデル動物で気管支肺胞洗浄液中のチトクロムc量を測定した結果を示す。The result of having measured the amount of cytochrome c in a bronchoalveolar lavage fluid with a model animal is shown. 特発性肺線維症患者の血清中チトクロムｃ量を測定した結果を示す。The result of having measured the amount of cytochrome c in serum of a patient with idiopathic pulmonary fibrosis is shown.

以下に、本発明の実施の形態について詳細に説明する。
本発明の検査方法は、肺傷害の検査方法であって、採取した体液を含む溶液中のチトクロムｃを定量することにより肺傷害を検査することを特徴とする。Hereinafter, embodiments of the present invention will be described in detail.
The test method of the present invention is a test method for lung injury, characterized by testing lung injury by quantifying cytochrome c in a solution containing a collected body fluid.

本発明において、肺傷害とは、肺胞細胞の傷害を意味する。肺疾患は、その種類により、肺傷害が現れる段階が異なるが、肺傷害を伴う場合が多い。従って、本発明は、肺傷害を伴う疾患、すなわち、肺傷害が現れる段階にある肺疾患の検査方法も提供する。進行段階により肺傷害の程度が異なる肺疾患であれば、本発明により肺疾患における段階が検査できる。   In the present invention, lung injury means injury of alveolar cells. Pulmonary diseases often involve lung injury, although the stage at which lung injury appears differs depending on the type of lung disease. Therefore, the present invention also provides a method for examining a disease associated with lung injury, that is, a lung disease in a stage where lung injury appears. If the lung disease has a different degree of lung injury depending on the progress stage, the stage in the lung disease can be examined according to the present invention.

ここで、肺疾患とは、肺癌，縦隔腫瘍，呼吸器感染症（肺炎，肺抗酸菌症，肺真菌症），慢性閉塞性肺疾患，間質性肺疾患（間質性肺炎），気管支喘息・アレルギー性肺疾患，自然気胸，サルコイドーシス，気管支拡張症，肺血栓塞栓症，睡眠時無呼吸症候群（外来簡易検査）等をいう。好ましくは、間質性肺疾患および急性呼吸窮迫症候群（ＡＲＤＳ）であり、特に好ましくは、急性呼吸窮迫症候群（ＡＲＤＳ）である。   Here, lung disease is lung cancer, mediastinal tumor, respiratory infection (pneumonia, pulmonary mycobacterial disease, pulmonary mycosis), chronic obstructive pulmonary disease, interstitial lung disease (interstitial pneumonia), Bronchial asthma / allergic lung disease, spontaneous pneumothorax, sarcoidosis, bronchiectasis, pulmonary thromboembolism, sleep apnea syndrome (outpatient simple examination), etc. Interstitial lung disease and acute respiratory distress syndrome (ARDS) are preferable, and acute respiratory distress syndrome (ARDS) is particularly preferable.

本発明の検査方法は、肺傷害が早い段階から現れる肺疾患や、肺傷害の有無による段階の判定が重要な肺疾患に適用することが好ましい。   The inspection method of the present invention is preferably applied to lung diseases that appear at an early stage of lung injury or lung diseases in which determination of the stage based on the presence or absence of lung injury is important.

採取した体液を含む溶液中のチトクロムｃを測定する方法としては、免疫化学的方法、電気泳動による方法、クロマトグラフィーによる方法等が考えられる。電気泳動による方法としては、ポリアクリルアミドゲル電気泳動を行ってチトクロムｃをバンドとして検出する方法、キャピラリー電気泳動でピークとして検出する方法等がある。また、クロマトグラフィーによる方法としては、高速液体クロマトグラフィーでピークとして検出する方法等がある。場合によっては感度を上げるために、蛍光標識することも許される。   As a method for measuring cytochrome c in a solution containing the collected body fluid, an immunochemical method, a method by electrophoresis, a method by chromatography, and the like can be considered. As a method by electrophoresis, there are a method of detecting cytochrome c as a band by performing polyacrylamide gel electrophoresis, a method of detecting as a peak by capillary electrophoresis, and the like. Further, as a method by chromatography, there is a method of detecting as a peak by high performance liquid chromatography. In some cases, fluorescent labeling is allowed to increase sensitivity.

体液を含む溶液としては、気管支肺胞洗浄液が挙げられるが、その他の体液（血液、血清、血漿若しくは脳脊髄液等）を含む溶液であってもよい。ここで、体液を含む溶液は、体液そのものであってもよい。
気管支肺胞洗浄液は、通常の方法により採取されたものを用いることができる。Examples of the solution containing body fluid include bronchoalveolar lavage fluid, but may also be a solution containing other body fluid (blood, serum, plasma, cerebrospinal fluid, etc.). Here, the solution containing the body fluid may be the body fluid itself.
The bronchoalveolar lavage fluid can be collected by a normal method.

チトクロムｃを測定する方法としては、感度及び簡便性から免疫化学的方法が好ましい。ここで免疫化学的方法とは、チトクロムｃに対する抗体を用いて、チトクロムｃを定量する方法である。免疫化学的方法としては、チトクロムｃを標識する競合法、抗体を標識するサンドイッチ法、抗体コートしたビーズの凝集を観察するラテックスビーズ法等、様々な方法があるが、チトクロムｃに対する抗体を用いた方法であれば、本発明の好ましい態様に含まれる。抗体はモノクローナル抗体でも、ポリクローナル抗体でも良い。また標識する方法にも、放射性同位元素による標識、電気化学発光する化合物による標識、蛍光標識、酵素標識、ビオチン標識等、様々な方法があるが、本発明はこれらの例に限られるものではない。   As a method for measuring cytochrome c, an immunochemical method is preferable from the viewpoint of sensitivity and simplicity. Here, the immunochemical method is a method for quantifying cytochrome c using an antibody against cytochrome c. There are various immunochemical methods such as a competitive method for labeling cytochrome c, a sandwich method for labeling an antibody, and a latex bead method for observing the aggregation of antibody-coated beads. An antibody against cytochrome c was used. If it is a method, it is contained in the preferable aspect of this invention. The antibody may be a monoclonal antibody or a polyclonal antibody. There are various labeling methods such as labeling with a radioisotope, labeling with an electrochemiluminescent compound, fluorescent labeling, enzyme labeling, biotin labeling, etc., but the present invention is not limited to these examples. .

本発明に使用される抗チトクロムｃ抗体は、チトクロムｃ（断片を含む）を検出できる抗体であれば特に制限はなく、ポリクローナル（抗血清）でもモノクローナル抗体でもよい。   The anti-cytochrome c antibody used in the present invention is not particularly limited as long as it can detect cytochrome c (including a fragment), and may be polyclonal (antiserum) or monoclonal antibody.

本発明で言うポリクローナル抗体（抗血清）あるいはモノクローナル抗体は、既存の一般的な製造方法によって製造することができ、モノクローナル抗体は、抗体産生細胞と自己抗体産生能のない骨髄腫系細胞（ミエローマ細胞）からハイブリドーマを調製し（ネイチャー(Nature)、第２５６巻、第４９５〜第４９７頁、１９７５年）、該ハイブリドーマをクローン化し、哺乳動物の免疫に用いた抗原に対して特異的親和性を示すモノクローナル抗体を産生するクローンを選択することによって製造することができる。   The polyclonal antibody (antiserum) or monoclonal antibody referred to in the present invention can be produced by an existing general production method. Monoclonal antibodies are antibody-producing cells and myeloma cells (myeloma cells) having no autoantibody-producing ability. ) (Nature, Vol. 256, 495-497, 1975), and the hybridoma is cloned and has a specific affinity for the antigen used for immunization of mammals. It can be produced by selecting clones that produce monoclonal antibodies.

またモノクローナル抗体の場合には、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤあるいはＩｇＥ等のいずれのアイソタイプを有するモノクローナル抗体をも包含する。好ましくは、ＩｇＧまたはＩｇＭである。   In the case of a monoclonal antibody, a monoclonal antibody having any isotype such as IgG, IgM, IgA, IgD, or IgE is also included. Preferably, it is IgG or IgM.

さらに、本発明における抗体には、抗体のフラグメントも含まれるものであり、この「抗体のフラグメント」とは、前述のようなモノクローナル抗体の一部分の領域を意味し、具体的にはF(ab')2 、Fab'、Fab 、Ｆｖ（variable fragment of antibody）、ｓＦｖ、ｄｓＦｖ（disulphide stabilised Fv）あるいはｄＡｂ（single domain antibody）などを意味する（エキスパート・オピニオン・オン・テラピューティック・パテンツ(Exp. Opin. Ther. Patents)，第６巻，第５号，第441〜456頁，1996年）。Furthermore, the antibody in the present invention includes an antibody fragment, and this “antibody fragment” means a partial region of the monoclonal antibody as described above, specifically, F (ab ′ ) 2 , Fab ′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv) or dAb (single domain antibody), etc. (Expert Opinion on Therapeutic Patents (Exp. Opin. Ther. Patents), Vol. 6, No. 5, pp. 441-456, 1996).

本発明の方法では、好ましくは酸性領域で抗チトクロムｃ抗体とチトクロムｃとを反応させる。本願明細書でいう酸性領域とは、抗体とチトクロムｃの結合に影響を与える体液中の妨害物質の影響が減弱するｐＨ領域である。ｐＨを低下させることにより妨害物質の影響を減弱させることができるが、同時に抗体とチトクロムｃの結合も弱くなるため、本発明の免疫化学的な測定におけるｐＨは、通常には、以下の条件を満たすｐＨに決められる。   In the method of the present invention, the anti-cytochrome c antibody and cytochrome c are preferably reacted in the acidic region. The acidic region as used herein is a pH region in which the influence of interfering substances in body fluids that affect the binding between an antibody and cytochrome c is attenuated. Although the influence of interfering substances can be attenuated by lowering the pH, at the same time, the binding between the antibody and cytochrome c is also weakened. Therefore, the pH in the immunochemical measurement of the present invention is usually as follows. The pH to be satisfied is determined.

１．緩衝液中で１０ｎｇ／ｍＬ、好ましくは１ｎｇ／ｍＬのチトクロムｃが定量可能な測定感度が得られる。
２．体液存在下での添加回収試験における回収率が７０％以上、好ましくは８０％以上、さらに好ましくは９０％以上得られる。1. A measurement sensitivity capable of quantifying 10 ng / mL, preferably 1 ng / mL of cytochrome c in the buffer is obtained.
2. A recovery rate in the addition recovery test in the presence of a body fluid is 70% or more, preferably 80% or more, and more preferably 90% or more.

妨害物質の影響および抗体とチトクロムｃの結合に及ぼすｐＨの効果は用いる抗体により異なるため、当該免疫化学的方法に用いるｐＨは、抗体毎に最適なｐＨを決めることができる。好ましくは、ｐＨ７以下、さらに好ましくはｐＨ３．０〜ｐＨ６．０、特に好ましくはｐＨ３．５〜ｐＨ５、その中でもさらに好ましくはｐＨ３．５〜ｐＨ４．５である。   Since the influence of the interfering substance and the effect of pH on the binding between the antibody and cytochrome c vary depending on the antibody used, the pH used for the immunochemical method can determine the optimum pH for each antibody. The pH is preferably 7 or less, more preferably pH 3.0 to pH 6.0, particularly preferably pH 3.5 to pH 5, and even more preferably pH 3.5 to pH 4.5.

以下に、免疫化学的な測定におけるｐＨを決める方法を具体的に示すが、ｐＨを決める方法は、これに限定されるものではない。   The method for determining the pH in the immunochemical measurement is specifically shown below, but the method for determining the pH is not limited to this.

１．測定感度へのｐＨの影響
ＢＳＡ等の適当な蛋白質、ＮａＣｌ等の適当な塩類、必要により適当な界面活性剤を含むｐＨ３〜ｐＨ７．５の緩衝液に、チトクロムｃを１〜１０００ｎｇ／ｍＬに希釈する。当該免疫化学的な測定法が２ステップサンドイッチ法の場合、希釈したチトクロムｃを固相化した抗チトクロムｃ抗体を反応させ、洗浄後、標識した抗チトクロムｃ抗体を加えて標識物質に対応した活性、放射性標識であれば放射活性、酵素標識であれば酵素活性により標識物質を検出する。1. Effect of pH on measurement sensitivity Cytochrome c is diluted to 1 to 1000 ng / mL in a pH 3 to pH 7.5 buffer solution containing an appropriate protein such as BSA, an appropriate salt such as NaCl, and an appropriate surfactant if necessary. To do. When the immunochemical measurement method is a two-step sandwich method, an anti-cytochrome c antibody obtained by immobilizing diluted cytochrome c is reacted, washed, and then labeled anti-cytochrome c antibody is added to obtain an activity corresponding to the labeling substance. If it is a radioactive label, the labeling substance is detected by radioactivity, and if it is an enzyme label, the labeling substance is detected by the enzyme activity.

また、当該免疫化学的な測定法が１ステップサンドイッチ法の場合、希釈したチトクロムｃと固相化した抗チトクロムｃ抗体、標識した抗チトクロムｃ抗体を加えて反応させ、標識物質に対応した活性、放射性標識であれば放射活性、酵素標識であれば酵素活性により標識物質を検出する。   In addition, when the immunochemical measurement method is a one-step sandwich method, diluted cytochrome c and immobilized anti-cytochrome c antibody and labeled anti-cytochrome c antibody are added and reacted, and the activity corresponding to the labeling substance, If it is a radioactive label, the labeling substance is detected by radioactivity.

10ｎｇ／ｍＬ、好ましくは1ｎｇ／ｍＬのチトクロムｃを含む検体から得られるシグナルが、チトクロムｃを含まない緩衝液のみの検体のシグナルと比較して充分に強ければ、当該pHは免疫化学的測定法に用いる緩衝液のpHの候補として挙げられる。   If the signal obtained from a sample containing cytochrome c at 10 ng / mL, preferably 1 ng / mL, is sufficiently strong compared to the signal from a sample containing only cytochrome c, the pH is determined by an immunochemical assay. Candidates for the pH of the buffer used in

２．添加回収へのpHの影響
免疫化学的測定法に用いる検体（例えば、気管支肺胞洗浄液）に、既知量のチトクロムｃを添加する。チトクロムｃを添加した検体、及び添加しなかった検体中のチトクロムｃ量を、当該免疫化学的な方法により測定し、理論値に対する測定値の比率を回収率とする。2. Effect of pH on addition and recovery A known amount of cytochrome c is added to a specimen (for example, bronchoalveolar lavage fluid) used for immunochemical measurement. The amount of cytochrome c in the sample to which cytochrome c was added and the sample to which cytochrome c was not added is measured by the immunochemical method, and the ratio of the measured value to the theoretical value is taken as the recovery rate.

回収率は、添加したチトクロムｃ量をＡ、チトクロムｃ未添加検体中のチトクロムｃ測定値をＢ、チトクロムｃ添加検体中のチトクロムｃ測定値をＣとして、
(1)測定値（Ｃ）／理論値（ＡとＢの加重平均）、
(2)チトクロムｃ添加による測定値の上昇値（Ｃ−Ｂ）／添加量（Ａ）、
の何れでも計算できる。The recovery rate is as follows: the amount of cytochrome c added is A, the measured value of cytochrome c in the sample not containing cytochrome c is B, and the measured value of cytochrome c in the sample added with cytochrome c is C.
(1) Measured value (C) / theoretical value (weighted average of A and B),
(2) Increase in measured value due to addition of cytochrome c (CB) / addition amount (A),
Either of these can be calculated.

回収率が70%以上、好ましくは80%以上、更に好ましくは90%以上の場合、当該pHは免疫化学的測定法に用いる緩衝液のpHの候補として挙げられる。   When the recovery rate is 70% or more, preferably 80% or more, and more preferably 90% or more, the pH can be listed as a candidate pH of a buffer used in an immunochemical measurement method.

当該免疫化学的な方法に用いる緩衝液のpHは１．測定感度へのpHの影響、２．添加回収へのpHの影響を勘案して決定する。緩衝液の種類は、酸性条件に調整し得る緩衝液であれば特に制限されないが、例えばクエン酸リン酸緩衝液などが挙げられる。   The pH of the buffer used for the immunochemical method is 1. 1. Effect of pH on measurement sensitivity Determined by taking into account the effect of pH on addition recovery. Although the kind of buffer solution will not be restrict | limited especially if it is a buffer solution which can be adjusted to acidic conditions, For example, a citrate phosphate buffer etc. are mentioned.

また、免疫化学的なチトクロムｃの測定に当たり、酸性領域の緩衝液を用いる方法は、固相化した抗体と検体中のチトクロムｃとを反応させる第１反応のみならず、第１反応で妨害物質が除去しきれなかった時に、第２反応（チトクロムｃと標識抗体を反応させる工程）に用いても有効である。
本態様の方法は、第１反応で酸性領域の緩衝液を用いる方法に限られるものではない。In addition, in the immunochemical measurement of cytochrome c, the method using an acidic region buffer is not limited to the first reaction in which the immobilized antibody and cytochrome c in the sample are reacted, but also in the first reaction. It is also effective to use for the second reaction (a step of reacting cytochrome c with a labeled antibody) when it has not been removed.
The method of this embodiment is not limited to the method using an acidic region buffer in the first reaction.

本態様の方法では、通常には、酸性条件で抗チトクロムｃ抗体とチトクロムｃとを反応させた後に、チトクロムｃの検出を行う。検出は、好ましくは洗浄を行った後、例えば、標識された2次抗体を反応させ、標識に応じた検出法により、検出する方法が挙げられる。例えば、ビオチン標識された2次抗体を用いる場合は、さらに、アビジン標識されたペルオキシダーゼやアルカリフォスファターゼなどの酵素を反応させた後、該酵素の基質を用いて発光又は発色により検出することができる。また、酵素標識された2次抗体、蛍光標識された2次抗体、ルテニウム標識された2次抗体などを用いてもよい。   In the method of this embodiment, usually, cytochrome c is detected after reacting the anti-cytochrome c antibody and cytochrome c under acidic conditions. The detection is preferably performed by washing and then reacting with a labeled secondary antibody and detecting by a detection method according to the label. For example, when a biotin-labeled secondary antibody is used, it can be detected by luminescence or color development using a substrate of the enzyme after further reacting with an enzyme such as avidin-labeled peroxidase or alkaline phosphatase. In addition, an enzyme-labeled secondary antibody, a fluorescently-labeled secondary antibody, a ruthenium-labeled secondary antibody, or the like may be used.

チトクロムｃを測定する免疫化学的方法の例としては、例えば下記の参考例に記載した方法で調製したルテニウム錯体標識抗チトクロムｃ抗体を用いる方法が挙げられる。   Examples of immunochemical methods for measuring cytochrome c include, for example, a method using a ruthenium complex-labeled anti-cytochrome c antibody prepared by the method described in the following Reference Example.

さらに本発明は、肺傷害検査用キットであって、採取した体液を含む溶液中のチトクロムcを定量するための試薬を含むことを特徴とするキット、及び、肺傷害を伴う肺疾患検査用キットであって、採取した体液を含む溶液中のチトクロムcを定量するための試薬を含むことを特徴とするキットに関する。本発明のキットは、好ましくは、体液を含む溶液中のチトクロムｃを免疫化学的に測定するための試薬、通常には、チトクロムｃ抗体を含む。本発明のキットは、さらに好ましくは、抗チトクロムｃ抗体とチトクロムｃの反応を酸性領域の緩衝液中で行うことを特徴とするキットである。例えば、キット付属の使用説明書に、酸性条件でチトクロムｃと抗チトクロムｃ抗体の反応を行う旨が記載されたキットが挙げられる。また、酸性に調整した反応用緩衝液を含むキットでもよい。本発明のキットは抗チトクロムｃ抗体の他に、2次抗体、チトクロムｃ標準液、希釈液、洗浄液、検出用の基質などを含むものでもよい。   Furthermore, the present invention provides a kit for testing lung injury, comprising a reagent for quantifying cytochrome c in a solution containing a collected body fluid, and a kit for testing lung disease accompanied by lung injury A kit comprising a reagent for quantifying cytochrome c in a solution containing a collected body fluid. The kit of the present invention preferably contains a reagent for immunochemically measuring cytochrome c in a solution containing body fluid, usually a cytochrome c antibody. More preferably, the kit of the present invention is characterized in that the reaction between the anti-cytochrome c antibody and cytochrome c is carried out in an acidic buffer solution. For example, a kit in which the reaction between cytochrome c and anti-cytochrome c antibody is performed under acidic conditions is described in the instruction manual attached to the kit. Moreover, the kit containing the buffer solution for reaction adjusted to acidity may be sufficient. In addition to the anti-cytochrome c antibody, the kit of the present invention may contain a secondary antibody, a cytochrome c standard solution, a diluent, a washing solution, a detection substrate, and the like.

本発明の検査キットは、通常には、体液を含む溶液中のチトクロムｃをチトクロムｃに対する抗体を用いて定量するための試薬を含む。この態様の試薬は、例えば、抗体として抗チトクロムｃ抗体を用いる以外は、通常のサンドイッチ法に用いられる試薬と同様の構成でよい。その一例としてサンドイッチ法によりチトクロムｃを測定する検査試薬は、例えば1)抗チトクロムｃ抗体コートカップ、抗チトクロムｃ抗体コートビーズなどの抗チトクロムｃ抗体コート固相、2)標識抗チトクロムｃ抗体、3)既知濃度のチトクロムｃ標準溶液、4)希釈液、5)洗浄液、を含有する試薬である。更に酵素標識であれば、6)発色基質、7)反応停止液が含まれてもよい。   The test kit of the present invention usually contains a reagent for quantifying cytochrome c in a solution containing body fluid using an antibody against cytochrome c. The reagent of this embodiment may have the same configuration as that of a reagent used in a normal sandwich method except that an anti-cytochrome c antibody is used as an antibody. As an example, test reagents for measuring cytochrome c by the sandwich method include, for example, 1) anti-cytochrome c antibody-coated solid phase such as anti-cytochrome c antibody-coated cup and anti-cytochrome c antibody-coated beads, 2) labeled anti-cytochrome c antibody, 3 A reagent containing a known concentration of cytochrome c standard solution, 4) a diluting solution, and 5) a washing solution. Furthermore, if it is an enzyme label, 6) a chromogenic substrate and 7) a reaction stop solution may be contained.

チトクロムｃの定量結果を指標として用いることにより、肺傷害の程度が検査される。例えばチトクロムｃの定量値が正常値と比較して高値であるときに、肺傷害と判定することができる。また、正常値上限（カットオフ値）を設定し（例えば、コントロール群の濃度の平均値+標準偏差の2倍）、それとの比較で肺傷害の有無を判定してもよい。肺傷害を伴う肺疾患の検査も肺傷害の程度の検査と同様に行うことができる。   By using the quantitative result of cytochrome c as an index, the degree of lung injury is examined. For example, lung injury can be determined when the quantitative value of cytochrome c is higher than the normal value. In addition, a normal value upper limit (cutoff value) may be set (for example, the average value of the concentration of the control group + twice the standard deviation), and the presence or absence of lung injury may be determined by comparison with it. The examination of the lung disease accompanied by lung injury can be performed in the same manner as the examination of the degree of lung injury.

以下に、具体的な例をもって本発明を示すが、本発明はこれに限られるものではない。％は、特記しない限り質量％である。   Hereinafter, the present invention will be described with specific examples, but the present invention is not limited thereto. % Means mass% unless otherwise specified.

［参考例１］抗チトクロムｃモノクローナル抗体の作製
ヒトチトクロムｃ（R&D Systems社）110μg/100μLと65 mMリン酸緩衝液pH 7.5に溶解した2 mg/mLオブアルブミン55μLを混合して、そこへ65 mMリン酸緩衝液pH 7.5で希釈した1 mMグルタルアルデヒド42μLを加え、室温で２時間撹拌した。次に、0.15 M NaClで４℃において４８時間透析し、等量のFCAとの混合物を作製し、BALB/Cマウス腹腔に0.1 mL免疫した。２週間おきに合計３回同様に免疫した。３回目の免疫の２週間後、マウスに生理食塩水に溶解したヒトチトクロムｃ50μg/100μLを尾静脈より静注した。３日後マウスから脾臓を摘出して、常法に従い、脾臓リンパ球をポリエチレングリコール法によりミエローマ細胞P3X63 Ag8U.1と細胞融合した。ヒトチトクロムｃを抗原としてスクリーニングを行い、ヒトチトクロムｃに対するモノクローナル抗体産生ハイブリドーマ（clone：27G9）を樹立した。[Reference Example 1] Preparation of anti-cytochrome c monoclonal antibody Human cytochrome c (R & D Systems) 110 μg / 100 μL and 2 mg / mL of albumin 55 μL dissolved in 65 mM phosphate buffer pH 7.5 were mixed, and 65 42 μL of 1 mM glutaraldehyde diluted with mM phosphate buffer pH 7.5 was added and stirred at room temperature for 2 hours. Next, the mixture was dialyzed against 0.15 M NaCl at 4 ° C. for 48 hours to prepare a mixture with an equal amount of FCA, and 0.1 mL of BALB / C mouse abdominal cavity was immunized. Immunization was performed in the same manner three times every two weeks. Two weeks after the third immunization, mice were intravenously injected with 50 μg / 100 μL of human cytochrome c dissolved in physiological saline through the tail vein. Three days later, the spleen was removed from the mouse, and spleen lymphocytes were fused with myeloma cells P3X63 Ag8U.1 by the polyethylene glycol method according to a conventional method. Screening was performed using human cytochrome c as an antigen to establish a monoclonal antibody-producing hybridoma (clone: 27G9) against human cytochrome c.

樹立したハイブリドーマをエスクロンSF-B培地（三光純薬社）で培養して増殖させ、BALB/Cマウス腹腔に接種した。１週間後、腹水を採取した。採取した腹水からプロテインＡを用いてIgGを精製し、抗チトクロムｃ抗体（27G9抗体）を得た。   The established hybridoma was grown by culturing in Escron SF-B medium (Sanko Junyaku Co., Ltd.) and inoculated into the peritoneal cavity of BALB / C mice. One week later, ascites was collected. IgG was purified from the collected ascites using protein A to obtain an anti-cytochrome c antibody (27G9 antibody).

［参考例２］抗チトクロムｃ抗体固相化ビーズの作製
抗チトクロムｃモノクローナル抗体（clone：2B5F8（R&D Systems社））を、0.15M NaClを含む0.1 M酢酸緩衝液（pH 4.2）で透析し、OD 280 nmが0.56になるよう0.15M NaClを含む0.1 M酢酸緩衝液（pH 4.2）で希釈した。希釈した抗体1.67 mLを、あらかじめ磁石を用いて0.15M NaClを含む0.1 M酢酸緩衝液（pH 4.2）3 mLで3回洗浄したビーズ（Dynabeads(登録商標) M-450 Epoxy, Dynal社）3.36 mL分と混合し、室温で１７時間撹拌した。次にビーズをブロッキングバッファー（50 mM Tris・HCl, 1% BSA, 0.15 M NaCl, 0.1% NaN3, pH7.5）3 mLで懸濁し、室温で７時間撹拌してビーズをブロッキングした。ブロッキングされたビーズを150mM Tris・HCl, 1% BSA, 0.3% トレハロース, 0.03容量% Tween 20, 0.3% NaN3, EDTA, pH7.5、3 mLで３回洗浄し、150mM Tris・HCl,3% BSA, 0.3% トレハロース, 0.03容量% Tween 20, 75μg/mLマウスIgG, 0.3% NaN3, pH7.5、12.5mLに懸濁した。これを使用時に濃度を調整して測定に用いた。[Reference Example 2] Preparation of anti-cytochrome c antibody-immobilized beads Anti-cytochrome c monoclonal antibody (clone: 2B5F8 (R & D Systems)) was dialyzed against 0.1 M acetate buffer (pH 4.2) containing 0.15 M NaCl, The solution was diluted with 0.1 M acetate buffer (pH 4.2) containing 0.15 M NaCl so that the OD 280 nm was 0.56. Beads (Dynabeads (registered trademark) M-450 Epoxy, Dynal) (3.36 mL) washed with 3 mL of 0.1 M acetate buffer (pH 4.2) containing 0.15M NaCl in advance using a magnet. And mixed for 17 hours at room temperature. Next, the beads were suspended in 3 mL of a blocking buffer (50 mM Tris · HCl, 1% BSA, 0.15 M NaCl, 0.1% NaN 3 , pH 7.5) and stirred at room temperature for 7 hours to block the beads. The blocked beads were washed 3 times with 150 mM Tris · HCl, 1% BSA, 0.3% trehalose, 0.03 vol% Tween 20, 0.3% NaN 3 , EDTA, pH 7.5, 3 mL, 150 mM Tris · HCl, 3% The suspension was suspended in BSA, 0.3% trehalose, 0.03 vol% Tween 20, 75 μg / mL mouse IgG, 0.3% NaN 3 , pH 7.5, 12.5 mL. This was used for measurement by adjusting the concentration at the time of use.

［参考例３］ルテニウム錯体標識抗チトクロムｃ抗体の作製
参考例１で作製した抗チトクロムｃモノクローナル抗体（27G9抗体）をPBSで透析し、抗体濃度を0.5 mg/mLから2 mg/mLの範囲に調製した。抗体1 mLにジメチルスルホキシドに10mg/mL濃度で溶解したルテニウム錯体（ruthenium(II) tris(bipyridyl)-N-hydroxysuccinimide, IGEN Corp. USA）を12.2μL加え室温で３０分撹拌した。次に2 M グリシンを50μL加え、室温で１０分撹拌した。それを、PBS-3（10 mMリン酸カリウム, 0.15M NaCl, 0.05% NaN3, pH 6）であらかじめ平衡化したSephadex G-25（GEヘルスケアバイオサイエンス社）カラム（1.5 cmφ x 30 cm）にアプライしてPBS-3で溶出し、1 mLでフラクション分取した。各フラクションのOD 280 nmを測定し、第１ピークのフラクションを集め、ルテニウム錯体標識抗チトクロムｃ抗体とした。抗体濃度をMicro BCA protein Assay kit（PIERCE社）を用いて測定した。[Reference Example 3] Preparation of ruthenium complex-labeled anti-cytochrome c antibody The anti-cytochrome c monoclonal antibody (27G9 antibody) prepared in Reference Example 1 was dialyzed with PBS to adjust the antibody concentration to a range of 0.5 mg / mL to 2 mg / mL. Prepared. 12.1 μL of ruthenium complex (ruthenium (II) tris (bipyridyl) -N-hydroxysuccinimide, IGEN Corp. USA) dissolved in dimethyl sulfoxide at a concentration of 10 mg / mL was added to 1 mL of the antibody and stirred at room temperature for 30 minutes. Next, 50 μL of 2 M glycine was added and stirred at room temperature for 10 minutes. Sephadex G-25 (GE Healthcare Biosciences) column (1.5 cmφ x 30 cm) pre-equilibrated with PBS-3 (10 mM potassium phosphate, 0.15 M NaCl, 0.05% NaN 3 , pH 6) And eluted with PBS-3, and fractions were collected at 1 mL. The OD 280 nm of each fraction was measured, and the first peak fraction was collected and used as a ruthenium complex-labeled anti-cytochrome c antibody. The antibody concentration was measured using Micro BCA protein Assay kit (PIERCE).

［参考例４］ヒトチトクロムｃ標準抗原の調製
チトクロムｃ標準抗原は、ヒトチトクロムｃ（R&D Systems社）を5% BSA、0.15 M NaCl, 0.1% NaN3を含む0.15 Mリン酸ナトリウム緩衝液pH 7.4で3000 ng/mL, 1000 ng/mL, 100 ng/mL, 10 ng/mL, 5 ng/mLに希釈して作製した。[Reference Example 4] Preparation of human cytochrome c standard antigen The cytochrome c standard antigen is human cytochrome c (R & D Systems) 0.15 M sodium phosphate buffer pH 7.4 containing 5% BSA, 0.15 M NaCl, 0.1% NaN 3. And diluted to 3000 ng / mL, 1000 ng / mL, 100 ng / mL, 10 ng / mL, and 5 ng / mL.

［実施例１］
急性呼吸窮迫（促迫）症候群、特発性肺線維症、非特異的間質性肺炎、持続性器質化肺炎、膠原病由来の間質性肺炎、過敏性肺炎、および、サルコイドーシスの患者、ならびに、健常者から気管支肺胞洗浄液を採取した。気管支肺胞洗浄液の採取方法は、1990年に厚生省特定疾患研究班により作成されたガイドラインに沿った。すなわち、滅菌生理食塩水150mLで気管支肺胞を洗浄し、回収した洗浄液をガーゼで漉して粘液を除去し、気管支肺胞洗浄液とした。[Example 1]
Patients with acute respiratory distress (impression) syndrome, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, persistent organizing pneumonia, interstitial pneumonia derived from collagen disease, hypersensitivity pneumonia, and sarcoidosis, and healthy Bronchoalveolar lavage fluid was collected from the subjects. The method for collecting bronchoalveolar lavage fluid was in accordance with the guidelines prepared by the Ministry of Health, Labor and Welfare specific disease research group in 1990. That is, the bronchoalveolar lavage was washed with 150 mL of sterilized physiological saline, and the collected lavage fluid was rinsed with gauze to remove mucus, thereby obtaining a bronchoalveolar lavage fluid.

検体希釈液（0.15M NaCl, 15mM EDTA, 2% Lipidure（登録商標)-BL802 (日本油脂社), 2% Lipidure(登録商標)-BL405 (日本油脂社), 0.1% NaN3を含む0.1M こはく酸緩衝液, pH4.0）をピコルミTM８２２０用反応管（三光純薬社）に200μL入れ、次に検体を20μL注入した。Sample diluent (0.15M NaCl, 15mM EDTA, 2% Lipidure (registered trademark) -BL802 (Nippon Yushi Co., Ltd.), 2% Lipidure (registered trademark) -BL405 (Nippon Yushi Co., Ltd.), 0.1M amber containing 0.1% NaN 3 200 μL of acid buffer (pH 4.0) was placed in a reaction tube for Picormi ™ 8220 (Sanko Junyaku Co., Ltd.), and then 20 μL of the sample was injected.

以下の測定は、電気化学発光酵素免疫測定機ピコルミTM８２２０（三光純薬社）を用いて行った。The following measurements were performed using an electrochemiluminescent enzyme immunoassay machine Picormi TM 8220 (Sanko Junyaku Co., Ltd.).

反応管に、1% BSA, 0.3% スクロース, 0.01 容量% Tween 20, 0.1% NaN3を含む0.15 M PBS, pH 7.5でビーズ濃度1mg/mLに調整した抗チトクロムｃ抗体固相化ビーズ25μLを加えて９分反応させ、ピコルミBF洗浄液（三光純薬社）350μLで2回洗浄後、1% BSA, 0.3%スクロース, 0.01 容量% Tween 20, 0.1% NaN3を含む0.15 M PBS, pH 7.5で0.5−１μg/mLに調整したルテニウム錯体標識抗チトクロムｃ抗体200μLを加えて９分反応させた。ピコルミBF洗浄液350μLで2回洗浄後、ピコルミ発光電解液（三光純薬社）を300μL加えて発光カウント値を計測した。Add 25 μL of anti-cytochrome c antibody-immobilized beads adjusted to a bead concentration of 1 mg / mL with 0.15 M PBS, pH 7.5 containing 1% BSA, 0.3% sucrose, 0.01 volume% Tween 20, 0.1% NaN 3 9 minutes reacted Te, washed twice with Picolumi BF washing solution (Sanko Junyaku Co.) 350μL, 1% BSA, 0.3 % sucrose, 0.01 volume% Tween 20, 0.15 M PBS containing 0.1% NaN 3, at pH 7.5 0.5 Ruthenium complex-labeled anti-cytochrome c antibody (200 μL) adjusted to −1 μg / mL was added and reacted for 9 minutes. After washing twice with 350 μL of Picormi BF cleaning solution, 300 μL of Picolmi luminescent electrolyte (Sanko Junyaku Co., Ltd.) was added and the luminescence count value was measured.

横軸にヒトチトクロムｃ標準抗原濃度、縦軸にヒトチトクロムｃ標準抗原のカウント値をプロットして標準曲線を描き、その標準曲線を基に、気管支肺洗浄液の発光カウント値からそれぞれに含まれるチトクロムｃ量を算出し、マンホイットニー順位和検定(Mann-Whitney U-test)により疾患群間における有意差を検討した。   A standard curve is drawn by plotting the human cytochrome c standard antigen concentration on the horizontal axis and the count value of human cytochrome c standard antigen on the vertical axis. Based on the standard curve, cytochrome contained in each of the luminescence count values of the bronchopulmonary lavage fluid The amount of c was calculated, and a significant difference between the disease groups was examined by a Mann-Whitney U-test.

その結果を図１に示す。図１によれば、非特異的間質性肺炎(NSIP)やコントロール(CONTROL)にはチトクロムｃ量の上昇が全く見られず、急性呼吸窮迫症候群(ARDS)、特発性肺線維症(IPF)、持続性器質化肺炎(COP)、膠原病由来の間質性肺炎(CVD-IP)、過敏性肺炎(HP)、サルコイドーシス(SA)ではチトクロムｃ量の上昇が見られた。特に急性呼吸窮迫症候群はチトクロムｃ量の上昇が顕著に見られた。チトクロムｃ量の上昇が見られない疾患は肺傷害を伴わない疾患であるか、又は、本実施例において気管支肺胞洗浄液を採取した患者では肺傷害が生じる段階に達していないと考えられる。   The result is shown in FIG. According to Fig. 1, non-specific interstitial pneumonia (NSIP) and control (CONTROL) showed no increase in cytochrome c content, acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF) Increased cytochrome c levels were observed in persistent organizing pneumonia (COP), interstitial pneumonia derived from collagen disease (CVD-IP), hypersensitivity pneumonia (HP), and sarcoidosis (SA). In particular, acute respiratory distress syndrome showed a marked increase in cytochrome c content. The disease in which no increase in cytochrome c level is observed is a disease that does not involve lung injury, or in the present example, it is considered that the stage in which lung injury has not occurred has been reached in the patient who collected bronchoalveolar lavage fluid.

次に、症例の疾患分類を詳細に検討した結果、急性呼吸窮迫症候群(ARDS)1例を過敏性肺炎(HP)に、特発性肺線維症(IPF)1例を急性呼吸窮迫症候群(ARDS)に変更した。更に、有意差検定の方法を分散分析（one way ＡＮＯＶＡ）および多重比較（post hoc analysis）に変更して検討した。   Next, as a result of detailed examination of the disease classification of cases, one case of acute respiratory distress syndrome (ARDS) was caused by hypersensitivity pneumonia (HP), one case of idiopathic pulmonary fibrosis (IPF) was caused by acute respiratory distress syndrome (ARDS) Changed to Furthermore, the method of the significant difference test was changed to an analysis of variance (one way ANOVA) and a multiple comparison (post hoc analysis).

その結果を図２および図３に示す。図２および図３によれば、非特異的間質性肺炎(NSIP)、持続性器質化肺炎(COP)およびコントロール(CONTROL)にはチトクロムｃ量の上昇が全く見られず、急性呼吸窮迫症候群(ARDS)、特発性肺線維症(IPF)、膠原病由来の間質性肺炎(CVD-IP)、過敏性肺炎(HP)、サルコイドーシス(SA)ではチトクロムｃ量の上昇が見られた（p<0.05）。特に急性呼吸窮迫症候群(ARDS)はチトクロムｃ量の上昇が顕著に見られた（p<0.0001）。チトクロムｃ量の上昇が見られない疾患は肺傷害を伴わない疾患であるか、又は、本実施例において気管支肺胞洗浄液を採取した患者では肺傷害が生じる段階に達していないと考えられる。   The results are shown in FIG. 2 and FIG. 2 and 3, according to nonspecific interstitial pneumonia (NSIP), persistent organizing pneumonia (COP) and control (CONTROL), no increase in cytochrome c level was observed, and acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF), interstitial pneumonia derived from collagen disease (CVD-IP), hypersensitivity pneumonia (HP), sarcoidosis (SA) showed increased cytochrome c levels (p <0.05). In particular, acute respiratory distress syndrome (ARDS) showed a marked increase in cytochrome c content (p <0.0001). It is considered that the disease in which no increase in cytochrome c level is observed is a disease that does not involve lung injury, or the stage in which lung injury has occurred has not been reached in the patient from whom bronchoalveolar lavage fluid was collected in this example.

［実施例２］
肺傷害の程度とチトクロムc量との関係をマウス実験動物モデルにおいて説明する。
ブレオマイシン(BLM)誘発肺傷害動物モデルは、ヒト急性肺傷害・特発性間質性肺炎のモデルとして広く使われている（Adamson IY. et al., American J. Pathol. 77:185-197, 1974）。７〜８週齢のC57BL/6雄性マウスにフェノバルビタールナトリウム（シェリング・プラウ社）を腹腔内に投与して麻酔し、体重1kgあたり、滅菌生理食塩水に溶解した3.0Uのブレオマイシン（日本化薬社）を気管内投与して、肺傷害動物モデルを作製した。ブレオマイシン非投与群、投与後７日群、投与後１４日群について、滅菌生理食塩水1mLで２回気管支肺胞内を洗浄して気管支肺胞洗浄液を採取した。気管支肺胞洗浄液中のチトクロムc量を測定した結果を図４に示す。[Example 2]
The relationship between the degree of lung injury and the amount of cytochrome c will be described in a mouse experimental animal model.
The bleomycin (BLM) -induced lung injury animal model is widely used as a model for human acute lung injury and idiopathic interstitial pneumonia (Adamson IY. Et al., American J. Pathol. 77: 185-197, 1974). ). 7-8 week old C57BL / 6 male mice were anesthetized by intraperitoneal administration of phenobarbital sodium (Schering-Plough) and 3.0 U bleomycin dissolved in sterile physiological saline per kg body weight (Nippon Kayaku) Was administered intratracheally to produce an animal model of lung injury. In the bleomycin non-administration group, the 7-day group after administration, and the 14-day group after administration, the bronchoalveolar lavage fluid was collected by washing the inside of the bronchoalveoli twice with 1 mL of sterile physiological saline. The results of measuring the amount of cytochrome c in the bronchoalveolar lavage fluid are shown in FIG.

以上のことより、チトクロムｃを測定することにより、肺傷害の程度が評価でき、また、肺傷害を伴う肺疾患、特に急性呼吸窮迫症候群を検査できることが示された。   From the above, it was shown that by measuring cytochrome c, the degree of lung injury can be evaluated, and lung diseases accompanying lung injury, particularly acute respiratory distress syndrome, can be examined.

［実施例３］
特発性肺線維症（ＩＰＦ）患者の血清中チトクロムc量を測定した。
検体希釈液（0.15M 塩化ナトリウム, 15mM EDTA, 2% Lipidure（登録商標)-BL802 (日本油脂社), 2% Lipidure(登録商標)-BL405 (日本油脂社), 0.1% NaN3を含む0.1M こはく酸緩衝液, pH4.0）をピコルミTM８２２０用反応管（三光純薬社）に200μL入れ、次に血清を20μL注入した。[Example 3]
Serum cytochrome c level was measured in patients with idiopathic pulmonary fibrosis (IPF).
Sample diluent (0.15M sodium chloride, 15mM EDTA, 2% Lipidure (registered trademark) -BL802 (Nippon Yushi Co., Ltd.), 2% Lipidure (registered trademark) -BL405 (Nippon Yushi Co., Ltd.), 0.1M amber containing 0.1% NaN3 200 μL of acid buffer (pH 4.0) was put into a reaction tube for Picormi ™ 8220 (Sanko Junyaku Co., Ltd.), and then 20 μL of serum was injected.

以下の測定は、電気化学発光酵素免疫測定機ピコルミTM８２２０（三光純薬社）を用いて行った。The following measurements were performed using an electrochemiluminescent enzyme immunoassay machine Picormi TM 8220 (Sanko Junyaku Co., Ltd.).

反応管に、1% BSA, 0.3% トレハロース, 10mM EDTA, 0.15M 塩化ナトリウム, 0.01 容量% Tween 20, 0.1% NaN3を含む50mM Tris・HCl, pH 7.5でビーズ濃度1mg/mLに調整した抗チトクロムｃ抗体固相化ビーズ25μLを加えて９分反応させ、ピコルミBF洗浄液（三光純薬社）350μLで2回洗浄後、1% BSA, 0.3%トレハロース, 0.15M 塩化ナトリウム, 0.01 容量% Tween 20, 0.1% NaN3を含む50 mM Tris・HCl, pH 7.5で0.5−1μg/mLに調整したルテニウム錯体標識抗チトクロムｃ抗体200μLを加えて９分反応させた。ピコルミBF洗浄液350μLで2回洗浄後、ピコルミ発光電解液（三光純薬社）を300μL加えて発光カウント値を計測した。Anti-cytochrome adjusted to a bead concentration of 1 mg / mL with 50 mM Tris · HCl, pH 7.5 containing 1% BSA, 0.3% trehalose, 10 mM EDTA, 0.15 M sodium chloride, 0.01 vol% Tween 20, 0.1% NaN 3 c Add 25 μL of antibody-immobilized beads and react for 9 minutes. After washing twice with 350 μL of Picormi BF washing solution (Sanko Junyaku Co., Ltd.), 1% BSA, 0.3% trehalose, 0.15 M sodium chloride, 0.01 vol% Tween 20, 200 μL of ruthenium complex-labeled anti-cytochrome c antibody adjusted to 0.5-1 μg / mL with 50 mM Tris · HCl, pH 7.5 containing 0.1% NaN 3 was added and reacted for 9 minutes. After washing twice with 350 μL of Picormi BF cleaning solution, 300 μL of Picolmi luminescent electrolyte (Sanko Junyaku Co., Ltd.) was added and the luminescence count value was measured.

横軸にヒトチトクロムｃ標準抗原濃度、縦軸にヒトチトクロムｃ標準抗原のカウント値をプロットして標準曲線を描き、その標準曲線を基に、気管支肺洗浄液の発光カウント値からそれぞれに含まれるチトクロムｃ量を算出した。   A standard curve is drawn by plotting the human cytochrome c standard antigen concentration on the horizontal axis and the count value of human cytochrome c standard antigen on the vertical axis, and based on the standard curve, cytochrome contained in each of the luminescence count values of bronchopulmonary lavage fluid c amount was calculated.

その結果を表１及び図５に示した。表１に血清を採取した日、最初の採取日からの日数、及びそれぞれのチトクロムｃ量を示した。疾患は臨床所見、検査所見により進行していることを確認し、チトクロムｃ量もそれに応じて上昇し、特に重篤な肺障害を伴った死亡例では、著明な高値を呈することが解った。   The results are shown in Table 1 and FIG. Table 1 shows the day of serum collection, the number of days from the first collection day, and the amount of each cytochrome c. It was confirmed that the disease was progressing from clinical findings and laboratory findings, and cytochrome c levels also increased accordingly, especially in death cases with severe pulmonary disorders, showing a markedly high value. .

Figure 2007145274
Figure 2007145274

以上のことより、チトクロムｃを測定することにより、肺傷害の程度が評価でき、また、肺傷害を伴う肺疾患を検査できることが示された。   From the above, it was shown that by measuring cytochrome c, the degree of lung injury can be evaluated and lung diseases associated with lung injury can be examined.

本発明によれば、肺傷害（肺胞細胞の傷害）の程度を容易に検査することができる。また、肺傷害を伴う肺疾患、特に間質性肺疾患や急性呼吸窮迫症候群の検査を行うことができる。   According to the present invention, the degree of lung injury (alveolar cell injury) can be easily examined. In addition, it is possible to examine pulmonary diseases associated with lung injury, particularly interstitial pulmonary diseases and acute respiratory distress syndrome.

Claims (34)

肺疾患における肺傷害の検査方法であって、採取した体液を含む溶液中のチトクロムｃを定量することにより肺傷害を検査することを特徴とする方法。 A method for examining lung injury in a lung disease, the method comprising examining lung injury by quantifying cytochrome c in a solution containing a collected body fluid. 肺傷害が間質性肺疾患における肺傷害である、請求項１記載の方法。 The method according to claim 1, wherein the lung injury is lung injury in interstitial lung disease. 肺傷害が急性呼吸窮迫症候群における肺傷害である、請求項１記載の方法。 The method of claim 1, wherein the lung injury is a lung injury in acute respiratory distress syndrome. 定量が免疫化学的方法による請求項１〜３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein the quantification is performed by an immunochemical method. 以下の工程を含む請求項４記載の方法。
（１）採取した体液中のチトクロムｃを免疫化学的方法により定量する工程
（２）定量した結果、正常値と比較して高値のときに肺傷害と判定する工程The method of Claim 4 including the following processes.
(1) A step of quantifying cytochrome c in the collected body fluid by an immunochemical method (2) A step of determining lung injury when the quantification results in a higher value compared to the normal value 免疫化学的方法が抗チトクロムｃ抗体とチトクロムｃの反応による方法である、請求項４又は５に記載の方法。 The method according to claim 4 or 5, wherein the immunochemical method is a method based on a reaction between an anti-cytochrome c antibody and cytochrome c. 抗チトクロムｃ抗体とチトクロムｃの反応を酸性領域の緩衝液中で行う、請求項６記載の方法。 The method according to claim 6, wherein the reaction between the anti-cytochrome c antibody and cytochrome c is carried out in an acidic buffer solution. 酸性領域がｐＨ３．０からｐＨ６．０である、請求項７記載の方法。 The method according to claim 7, wherein the acidic region is pH 3.0 to pH 6.0. 体液を含む溶液が気管支肺胞洗浄液である請求項１〜８のいずれか１項に記載の方法。 The method according to any one of claims 1 to 8, wherein the solution containing body fluid is bronchoalveolar lavage fluid. 肺傷害検査用キットであって、採取した体液を含む溶液中のチトクロムcを定量するための試薬を含むことを特徴とするキット。 A kit for testing lung injury, comprising a reagent for quantifying cytochrome c in a solution containing a collected body fluid. 肺傷害が間質性肺疾患における肺傷害である、請求項１０記載のキット。 The kit according to claim 10, wherein the lung injury is lung injury in interstitial lung disease. 肺傷害が急性呼吸窮迫症候群における肺傷害である、請求項１０記載のキット。 The kit according to claim 10, wherein the lung injury is lung injury in acute respiratory distress syndrome. 定量が免疫化学的方法による請求項１０〜１２のいずれか１項に記載のキット。 The kit according to any one of claims 10 to 12, wherein the quantification is performed by an immunochemical method. 免疫化学的方法が抗チトクロムｃ抗体とチトクロムｃの反応による方法である、請求項１３に記載のキット。 The kit according to claim 13, wherein the immunochemical method is a method based on a reaction between an anti-cytochrome c antibody and cytochrome c. 抗チトクロムｃ抗体とチトクロムｃの反応を酸性領域の緩衝液中で行うための緩衝液を含む、請求項１４記載のキット。 The kit of Claim 14 containing the buffer solution for performing reaction of an anti-cytochrome c antibody and cytochrome c in the buffer solution of an acidic region. 酸性領域がｐＨ３．０からｐＨ６．０である、請求項１５記載のキット。 The kit according to claim 15, wherein the acidic region is pH 3.0 to pH 6.0. 体液を含む溶液が気管支肺胞洗浄液である請求項１０〜１６のいずれか１項に記載のキット。 The kit according to any one of claims 10 to 16, wherein the solution containing body fluid is bronchoalveolar lavage fluid. 肺傷害を伴う肺疾患を検査する方法であって、採取した体液を含む溶液中のチトクロムｃを定量することにより肺傷害を伴う肺疾患を検査することを特徴とする方法。 A method for examining a pulmonary disease associated with lung injury, wherein the pulmonary disease associated with a lung injury is examined by quantifying cytochrome c in a solution containing a collected body fluid. 肺疾患が間質性肺疾患である、請求項１８記載の方法。 The method of claim 18, wherein the lung disease is interstitial lung disease. 肺疾患が急性呼吸窮迫症候群である、請求項１８記載の方法。 19. The method of claim 18, wherein the pulmonary disease is acute respiratory distress syndrome. 定量が免疫化学的方法による請求項１８〜２０のいずれか１項に記載の方法。 21. The method according to any one of claims 18 to 20, wherein the quantification is performed by an immunochemical method. 以下の工程を含む請求項２１に記載の方法。
（１）採取した体液中のチトクロムｃを免疫化学的方法により定量する工程
（２）定量した結果、正常値と比較して高値のときに肺傷害を伴う肺疾患と判定する工程The method of claim 21, comprising the following steps.
(1) A step of quantifying cytochrome c in the collected body fluid by an immunochemical method (2) A step of determining a lung disease associated with lung injury when the quantification results in a higher value compared to the normal value 免疫化学的方法が抗チトクロムｃ抗体とチトクロムｃの反応による方法である、請求項２１又は２２記載の方法。 The method according to claim 21 or 22, wherein the immunochemical method is a method based on a reaction between an anti-cytochrome c antibody and cytochrome c. 抗チトクロムｃ抗体とチトクロムｃの反応を酸性領域の緩衝液中で行う、請求項２３記載の方法。 The method according to claim 23, wherein the reaction between the anti-cytochrome c antibody and cytochrome c is carried out in an acidic buffer solution. 酸性領域がｐＨ３．０からｐＨ６．０である、請求項２４記載の方法。 25. The method of claim 24, wherein the acidic region is pH 3.0 to pH 6.0. 体液を含む溶液が気管支肺胞洗浄液である請求項１８〜２５のいずれか１項に記載の方法。 The method according to any one of claims 18 to 25, wherein the solution containing a body fluid is bronchoalveolar lavage fluid. 肺傷害を伴う肺疾患の検査用キットであって、採取した気管支肺胞洗浄液中のチトクロムcを定量するための試薬を含むことを特徴とするキット。 A kit for testing a lung disease accompanied by lung injury, comprising a reagent for quantifying cytochrome c in a collected bronchoalveolar lavage fluid. 肺疾患が間質性肺疾患である、請求項２７記載のキット。 28. The kit according to claim 27, wherein the lung disease is interstitial lung disease. 肺疾患が急性呼吸窮迫症候群である、請求項２７記載のキット。 28. The kit of claim 27, wherein the lung disease is acute respiratory distress syndrome. 定量が免疫化学的方法による請求項２７〜２９のいずれか１項に記載のキット。 30. The kit according to any one of claims 27 to 29, wherein the quantification is performed by an immunochemical method. 免疫化学的方法が抗チトクロムｃ抗体とチトクロムｃの反応による方法である、請求項３０記載のキット。 The kit according to claim 30, wherein the immunochemical method is a method based on a reaction between an anti-cytochrome c antibody and cytochrome c. 抗チトクロムｃ抗体とチトクロムｃの反応を酸性領域の緩衝液中で行うための緩衝液を含む、請求項３１記載のキット。 32. The kit according to claim 31, comprising a buffer for carrying out the reaction between the anti-cytochrome c antibody and cytochrome c in an acidic buffer. 酸性領域がｐＨ３．０からｐＨ６．０である、請求項３２記載のキット。 The kit according to claim 32, wherein the acidic region is pH 3.0 to pH 6.0. 体液を含む溶液が気管支肺胞洗浄液である請求項２７〜３３のいずれか１項に記載のキット。 The kit according to any one of claims 27 to 33, wherein the solution containing a body fluid is bronchoalveolar lavage fluid.
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