JP6893406B2 - Testing method for progress of NASH and NAFLD and testing method for inflammatory diseases and cancer - Google Patents
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Description
本発明は、多量体のガレクチン-３結合蛋白質(Ｍａｃ−２ｂｐ)を用いたＮＡＳＨやＮＡＦＬＤの進展度の検査方法及び炎症疾患や癌の検出方法に関する。 The present invention relates to a method for examining the degree of progression of NASH and NAFLD using a multimeric galectin-3 binding protein (Mac-2bp), and a method for detecting inflammatory diseases and cancers.
肝臓の組織中、脂肪滴を伴う肝細胞が３０％以上認められる場合、脂肪肝と診断される。脂肪肝はこれまでアルコールの摂取を原因とするものが多かったが、メタボリックシンドローム、肥満、糖尿病といった生活習慣病の患者でも認められるようになってきた。この中でも、飲酒歴はないがアルコール性脂肪肝に類似した脂肪肝がみられる病態が非アルコール性脂肪性肝疾患（ｎｏｎａｌｃｏｈｏｌｉｃ ｆａｔｔｙ ｌｉｖｅｒ ｄｉｓｅａｓｅ：ＮＡＦＬＤ）と呼ばれている。ＮＡＦＬＤの患者は国内で約１０００万人いると推定されている。ＮＡＦＬＤは、組織診断において肝細胞の脂肪沈着のみを認める非アルコール性脂肪肝（ｎｏｎａｌｃｏｈｏｌｉｃ ｆａｔｔｙ ｌｉｖｅｒ：ＮＡＦＬ）と、脂肪化に壊死・炎症や線維化を伴う進行性の非アルコール性脂肪肝炎（ｎｏｎａｌｃｏｈｏｌｉｃ ｓｔｅａｔｏｈｅｐａｔｉｔｉｓ：ＮＡＳＨ）に分けられる。ＮＡＳＨはＮＡＦＬＤの重症型で患者の１０〜２０％を占める。治療介入が無い場合、ＮＡＳＨ患者は５〜１０年で５〜２０％の症例で肝硬変へ進行する（非特許文献１）。 Fatty liver is diagnosed when 30% or more of hepatocytes with lipid droplets are found in the liver tissue. Fatty liver has been mostly caused by alcohol intake, but it is now being recognized in patients with lifestyle-related diseases such as metabolic syndrome, obesity, and diabetes. Among these, a condition in which fatty liver similar to alcoholic fatty liver is observed although there is no history of drinking is called non-alcoholic fatty liver disease (NAFLD). It is estimated that there are about 10 million patients with NAFLD in the country. NAFLD includes non-alcoholic fatty liver (NAFL), which shows only fat deposition of hepatic cells in histological diagnosis, and progressive non-alcoholic steatohepatitis, which is accompanied by necrosis / inflammation and fibrosis in fattening. : NASH). NASH is a severe form of NAFLD and accounts for 10-20% of patients. In the absence of therapeutic intervention, NASH patients progress to cirrhosis in 5 to 20% of cases in 5 to 10 years (Non-Patent Document 1).
ＮＡＳＨの診断における標準の診断方法は、肝生検である。病理学的診断では、肝細胞の大滴性脂肪化に加えて、炎症を伴う肝細胞の風船様変性を認めるものをＮＡＳＨとしている。肝生検は肝臓の一部を採取して観察することから、病理医によって診断結果にばらつきが生じうる。さらに肝生検の費用は高く、侵襲性も高いため、複数回実施することは難しく、全ての症例に肝生検を実施することは不可能である。そのため、非侵襲的な診断法の開発が望まれている。 The standard diagnostic method for diagnosing NASH is liver biopsy. In the pathological diagnosis, NASH is defined as those in which hepatocyte balloon-like degeneration accompanied by inflammation is observed in addition to large drop fattening of hepatocytes. Since a liver biopsy takes a part of the liver and observes it, the diagnosis result may vary depending on the pathologist. In addition, the cost and invasiveness of liver biopsy makes it difficult to perform multiple times and it is not possible to perform liver biopsy in all cases. Therefore, the development of a non-invasive diagnostic method is desired.
現時点ではＮＡＳＨとＮＡＦＬを鑑別できる確立された血液検査マーカーは存在しない。ＡＳＴ、ＡＬＴ、ＡＳＴ／ＡＬＴ ｒａｔｉｏ（ＡＡＲ）、血清フェリチン、ヒアルロン酸、ＩＶ型コラーゲン７Ｓ、高感度ＣＲＰ、ＨＯＭＡ−ＩＲなどの有用性が期待されているが、国内外で、多数例で十分に妥当性が確認されたものは少ない。アポトーシスのマーカーであるサイトケラチン１８断片（ＣＫ１８ ｆｒａｇｍｅｎｔ）はその有用性が期待されるが、現在のところ一般臨床検査値として普及していない。 At this time, there are no established blood test markers that can distinguish NASH from NAFL. The usefulness of AST, ALT, AST / ALT ratio (AAR), serum ferritin, hyaluronic acid, type IV collagen 7S, high-sensitivity CRP, HOMA-IR, etc. is expected, but many cases are sufficient in Japan and overseas. Few have been confirmed to be valid. Cytokeratin 18 fragment (CK18 fragment), which is a marker of apoptosis, is expected to be useful, but it is not widely used as a general clinical laboratory test value at present.
又、特許文献１には糖蛋白質としてＭａｃ−２ｂｐを測定することにより肝疾患の検査が可能ということが記載されている。この検査は、糖鎖変化を有したＭａｃ−２ｂｐを測定し、肝線維化進展の診断に利用されている。ＮＡＳＨは肝線維化を有さずとも肝細胞の風船様変性があればＮＡＳＨと診断されるため、ＮＡＳＨの診断とは相関しない。
Further,
又、非特許文献２には血中Ｍａｃ−２ｂｐ濃度のＥＬＩＳＡ法での測定が、ＮＡＳＨ鑑別のバイオマーカーとしてＣＫ１８ ｆｒａｇｍｅｎｔよりも優れているということが記載されている。しかし、平均値では有意な差があるものの、ＮＡＳＨであるのにも関わらずＭａｃ−２ｂｐ濃度が健常人と同レベルの患者や、Ｍａｃ−２ｂｐ濃度が高値の健常人が存在する。
Further,
本発明は、従来よりも高い精度でＮＡＳＨを診断できるＮＡＳＨやＮＡＦＬＤの進展度の検査方法及び炎症疾患や癌の検出方法を提供することを課題とする。 An object of the present invention is to provide a method for examining the degree of progression of NASH and NAFLD and a method for detecting inflammatory diseases and cancers, which can diagnose NASH with higher accuracy than before.
本発明者等は前記課題を解決すべく鋭意研究の結果、分子量９００ｋ以上のＭａｃ−２ｂｐを測定する事により、ＮＡＳＨやＮＡＦＬＤの進展度及び炎症疾患や癌の検出をより明確に診断することを見出し、本発明を完成させるに至った。 As a result of diligent research to solve the above problems, the present inventors have decided to more clearly diagnose the degree of progression of NASH and NAFLD and the detection of inflammatory diseases and cancers by measuring Mac-2bp having a molecular weight of 900 k or more. We have found and completed the present invention.
すなわち本発明は、
（１）生体試料中の分子量９００ｋ以上として検出されるＭａｃ−２ｂｐ濃度を検出することを特徴とするＮＡＳＨやＮＡＦＬＤの進展度を判定するための検査方法。
（２）前記生体試料は血清試料又は血漿試料である、前記ＮＡＳＨやＮＡＦＬＤの進展度を判定するための検査方法。
That is, the present invention
(1) An inspection method for determining the degree of progress of NASH or NAFLD, which comprises detecting the Mac-2bp concentration detected as having a molecular weight of 900 k or more in a biological sample.
(2) A test method for determining the degree of progression of NASH or NAFLD, wherein the biological sample is a serum sample or a plasma sample.
（３）Ｍａｃ−２ｂｐ濃度の検出が生体試料から分子量９００ｋ以上として検出されるＭａｃ−２ｂｐを分離した後に実施されることを特徴とする前記ＮＡＳＨやＮＡＦＬＤの進展度を判定するための検査方法。 (3) A test method for determining the degree of progress of NASH or NAFLD, wherein the detection of Mac-2bp concentration is performed after separating Mac-2bp detected as having a molecular weight of 900 k or more from a biological sample.
（４）Ｍａｃ−２ｂｐ濃度の検出を分子量９００ｋ以上のＭａｃ−２ｂｐに対して特異的かつ選択的な抗体を使用して実施されることを特徴とする前記ＮＡＳＨやＮＡＦＬＤの進展度を判定するための検査方法。 (4) To determine the degree of progress of the NASH or NAFLD, which is characterized in that the detection of the Mac-2bp concentration is carried out using an antibody specific and selective for Mac-2bp having a molecular weight of 900 k or more. Inspection method.
（５）測定法がラテックス凝集比濁法であることを特徴とする前記ＮＡＳＨ及びＮＡＦＬＤの進展度を判定するための方法。 (5) A method for determining the degree of progress of NASH and NAFLD, wherein the measuring method is a latex agglutination turbidimetry method.
（６）分子量９００ｋ以上のＭａｃ−２ｂｐが１０量体以上のＭａｃ−２ｂｐである前記ＮＡＳＨ及びＮＡＦＬＤの進展度を判定するための方法。 (6) A method for determining the degree of progress of the NASH and NAFLD in which Mac-2bp having a molecular weight of 900 k or more is Mac-2bp having a molecular weight of a dimer or more.
（７）分子量９００ｋ以上の物質がＭａｃ−２ｂｐの単量体や副量体が結合した蛋白である前記ＮＡＳＨ及びＮＡＦＬＤの進展度を判定するための検査方法。 (7) A test method for determining the degree of progress of the NASH and NAFLD, which are proteins in which a substance having a molecular weight of 900 k or more is a protein to which a monomer or a submer of Mac-2bp is bound.
（８）生体試料中の分子量９００ｋ以上として検出されるＭａｃ−２ｂｐ濃度を検出することを特徴とする炎症疾患及び癌を検出する方法。 (8) A method for detecting an inflammatory disease and cancer, which comprises detecting a Mac-2bp concentration detected as having a molecular weight of 900 k or more in a biological sample.
（９）検出する炎症疾患及び癌が肝炎、肝硬変及び肝癌、前立腺癌であることを特徴とする前記炎症疾患及び癌を検出する方法
に関する。
(9) The present invention relates to a method for detecting the inflammatory disease and cancer, which is characterized in that the inflammatory disease and cancer to be detected are hepatitis, liver cirrhosis and liver cancer, and prostate cancer.
分子量９００ｋ以上のＭａｃ−２ｂｐを測定する事により、ＮＡＳＨやＮＡＦＬＤの進展度及び炎症疾患や癌の検出をより明確に判定することができる。 By measuring Mac-2bp having a molecular weight of 900 k or more, the degree of progression of NASH and NAFLD and the detection of inflammatory diseases and cancer can be determined more clearly.
生体試料は、対象から排泄、採取した試料をさす。種類は特に限定されないが、例えば、排泄物、腹水、咽頭ぬぐい液、涙液、脳脊髄液、血液、血清、血漿などの体液、組織、細胞などが挙げられる。好ましい生体試料は、血液試料（全血）である。さらに好ましい生体試料は、血清試料又は血漿試料である。より好ましい生体試料は、血清試料である。前記生体試料の採取方法に、特に制限はなく、常法により採取することができる。例えば、血液試料の場合、静脈採血や動脈採血により採取される。また、前記血液試料の採取される部位としても、特に制限はなく、体のどの部位から採取された血液であってもよい。 A biological sample refers to a sample excreted or collected from a subject. The type is not particularly limited, and examples thereof include excrement, ascites, pharyngeal swab, tears, cerebrospinal fluid, blood, serum, body fluids such as plasma, tissues, and cells. A preferred biological sample is a blood sample (whole blood). More preferred biological samples are serum or plasma samples. A more preferred biological sample is a serum sample. The method for collecting the biological sample is not particularly limited, and the biological sample can be collected by a conventional method. For example, in the case of a blood sample, it is collected by intravenous blood sampling or arterial blood sampling. The site from which the blood sample is collected is not particularly limited, and blood may be collected from any part of the body.
本発明において、「対象」とは、哺乳動物（例えば、ヒト、サル、イヌ、ネコ、ウサギ、ウシ、ウマ、ヒツジ、ヤギ、ブタ、シカなど）であれば特に限定されないが、好ましくはヒトである。 In the present invention, the "subject" is not particularly limited as long as it is a mammal (for example, human, monkey, dog, cat, rabbit, cow, horse, sheep, goat, pig, deer, etc.), but is preferably human. is there.
本発明において、分子量９００ｋ以上のＭａｃ−２ｂｐとは、Ｍａｃ−２ｂｐの単量体や重合体が生体物質と結合した蛋白質又はＭａｃ−２ｂｐのみが重合した蛋白質である。Ｍａｃ−２ｂｐのみが重合する場合、１０量体以上となると分子量が９００ｋ以上となる。 In the present invention, Mac-2bp having a molecular weight of 900 k or more is a protein in which a monomer or polymer of Mac-2bp is bound to a biological substance or a protein in which only Mac-2bp is polymerized. When only Mac-2bp is polymerized, the molecular weight becomes 900 k or more when it becomes a dimer or more.
本発明において分子量９００ｋ以上のＭａｃ−２ｂｐ検査の検出方法は、定量可能な方法であればよく、特に限定されない。例えば、免疫学的手法による方法であり、間接蛍光抗体法、二重免疫拡散法、ラジオイムノアッセイ法、ウエスタンブロッティング法、ＥＬＩＳＡ法、ラテックス法、受身赤血球凝集反応、免疫沈降法、免疫ブロット法やイムノクロマト法など公知の手法で実施することができる。検出の際に使用し得る標識物質、測定機器などは、その目的・手段に適用できるものであれば特に限定されず、公知のものを適用することができる。 In the present invention, the detection method for the Mac-2bp test having a molecular weight of 900 k or more is not particularly limited as long as it is a quantifiable method. For example, it is an immunological method, such as indirect immunofluorescence method, double immunodiffusion method, radioimmunoassay method, Western blotting method, ELISA method, latex method, passive hemagglutination reaction, immunoprecipitation method, immunoprecipitation method and immunochromatography. It can be carried out by a known method such as a method. The labeling substance, measuring device, and the like that can be used for detection are not particularly limited as long as they can be applied to the purpose and means, and known substances can be applied.
本発明の検出手段は、比色法（染色法・着色コロイドや着色ラテックスなど含む）、蛍光法や発光法（化学・生物）などを挙げることができる。 Examples of the detection means of the present invention include a colorimetric method (including a dyeing method / colored colloid and colored latex), a fluorescence method and a light emitting method (chemistry / biology).
分子量９００ｋ以上のＭａｃ−２ｂｐを分離する手段は、分子量毎に分画できるものであればよく、分子篩機能を有するゲル、膜や樹脂などを挙げることができる。
ポリアクリルアミド（やアガロース）などゲル電気泳動やキャピラリー電気泳動、分離形状はフィルター分離やカラム分離などを挙げることができる。
The means for separating Mac-2bp having a molecular weight of 900 k or more may be any means as long as it can be fractionated for each molecular weight, and examples thereof include gels, membranes and resins having a molecular sieve function.
Gel electrophoresis such as polyacrylamide (or agarose) and capillary electrophoresis, and separation shapes include filter separation and column separation.
分子量９００ｋ以上のＭａｃ−２ｂｐを分離して本発明の検査方法を実施する場合は、分子量９００ｋ以上のＭａｃ−２ｂｐを分離後連続的に実施されることが望ましい。例えば、イムノクロマト法の様な方法が例として挙げられる。 When the test method of the present invention is carried out by separating Mac-2bp having a molecular weight of 900 k or more, it is desirable that Mac-2bp having a molecular weight of 900 k or more is continuously carried out after separation. For example, a method such as the immunochromatography method can be mentioned as an example.
分子量９００ｋ以上のＭａｃ−２ｂｐに対して特異的かつ選択的な抗体は、通常の免疫染色やＥＬＩＳＡ法で使用可能な抗体から選択しても良い。ポリクローナル抗体やモノクローナル抗体などで良く、例えば、Ｍａｃ−２ｂｐの糖鎖を認識する抗体であっても良い（例えば特許第５０３１９２８号に記載の抗体）。この他、ＩＢＬ社から入手可能な８Ａ２、１１Ａ１、１２Ａ１、３１Ａ１、４７Ａ１、６７Ａ１、６９Ａ１が挙げられる。本発明においては検出方法に合わせて、１種類又は複数の抗体を用いる。 Antibodies specific and selective for Mac-2bp having a molecular weight of 900 k or more may be selected from antibodies that can be used by ordinary immunostaining or ELISA. It may be a polyclonal antibody, a monoclonal antibody, or the like, and may be, for example, an antibody that recognizes the sugar chain of Mac-2bp (for example, the antibody described in Japanese Patent No. 5031928). In addition, 8A2, 11A1, 12A1, 31A1, 47A1, 67A1, 69A1 available from IBL can be mentioned. In the present invention, one or more antibodies are used according to the detection method.
以下実施例により本発明を更に詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to Examples.
[参考例１]
ＥＬＩＳＡによるＭａｃ−２ｂｐ濃度測定
株式会社免疫生物研究所（ＩＢＬ）社製のＨｕｍａｎ Ｍａｃ−２ ｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ（Ｍａｃ−２ｂｐ）Ａｓｓａｙ Ｋｉｔを用いて、健常人の血清９例、ＮＡＳＨ患者の血清９例のＭａｃ−２ｂｐ濃度の測定を行った。測定方法は製品データシートに従って行った。
結果を図１に示した。平均値では、健常人が０．８６μｇ／ｍｌに対してＮＡＳＨ患者が１．８１μｇ／ｍｌと有意な増加が認められた。しかし、ＮＡＳＨ患者の低値の検体（１．１８μｇ／ｍｌ）よりも高い値を示した健常人の検体が２検体（１．２８μｇ／ｍｌ、１．３４μｇ／ｍｌ）存在した。この結果より、健常人とＮＡＳＨ患者で値の重なりが認められた。
[Reference example 1]
Measurement of Mac-2bp concentration by ELISA Using Human Mac-2 binding protein (Mac-2bp) Assay Kit manufactured by Immunobiological Research Institute Co., Ltd. (IBL), 9 sera of healthy subjects and 9 sera of NASH patients The Mac-2bp concentration was measured. The measurement method was performed according to the product data sheet.
The results are shown in FIG. The average value was 0.86 μg / ml for healthy subjects and 1.81 μg / ml for NASH patients, showing a significant increase. However, there were two samples (1.28 μg / ml, 1.34 μg / ml) of healthy subjects that showed higher values than the low values of NASH patients (1.18 μg / ml). From this result, overlapping values were observed in healthy subjects and NASH patients.
[参考例２]
全自動免疫測定装置によるＭａｃ−２結合蛋白糖鎖修飾異性体（Ｍ２ＢＰＧｉ）濃度測定
シスメックス株式会社製の肝臓の線維化検査用試薬（ＨＩＳＣＬ Ｍ２ＢＰＧｉ試薬）を用いて、健常人の血清９例ＮＡＳＨ患者の血清９例のＭａｃ−２ｂｐ濃度の測定を行った。
結果を図２に示した。平均値では、健常人が０．５６Ｃｕｔ−Ｏｆｆ Ｉｎｄｅｘ（Ｃ．Ｏ．Ｉ．）に対してＮＡＳＨ患者が１．００Ｃ．Ｏ．Ｉ．と増加しているものの統計的な有意な差は認められなかった。
[Reference example 2]
Measurement of Mac-2 binding protein sugar chain modified isomer (M2BPGi) concentration using a fully
The results are shown in FIG. On average, healthy subjects had 0.56 Cut-Off Index (COI) and NASH patients had 1.00 C.I. O. I. However, no statistically significant difference was observed.
[比較例１及び実施例１]ウエスタンブロッティング法を用いた分子量９００ｋ以上のＭａｃ−２ｂｐ測定
ＮＡＳＨ鑑別のバイオマーカーである分子量９００ｋ以上のＭａｃ−２ｂｐについて、Ｎａｔｉｖｅ ＰＡＧＥ電気泳動及びウエスタンブロッティング法を活用し、ＮＡＳＨの検出を実施した例を以下に示す。
[Comparative Example 1 and Example 1] Measurement of Mac-2bp having a molecular weight of 900 k or more using a Western blotting method For Mac-2bp having a molecular weight of 900 k or more, which is a biomarker for NASH differentiation, Native PAGE electrophoresis and Western blotting are utilized. , An example of detecting NASH is shown below.
スタンダードとしてＩＢＬ社製のＭａｃ−２ｂｐを用いた。 Mac-2bp manufactured by IBL was used as a standard.
まず、以下の条件でＮａｔｉｖｅ ＰＡＧＥ電気泳動を行った。
（１）ゲル
４−２０％ Ｎｏｎ−ＳＤＳ ＰＡＧＥ ｍｉｎｉ １ｍｍ ８ｗｅｌｌ（ＴＥＦＣＯ社製）
（２）電気泳動層
セイフティーセルミニＳＴＣ−８０８（ＴＥＦＣＯ社製）
（３）泳動バッファー
０．０２５Ｍトリスヒドロキシメチルアミノメタン ＋ ０．２Ｍグリシン
（４）サンプル希釈液
１０％グリセロール ＋ ０．０２％ブロムフェノールブルー
（５）サンプル希釈倍率
５倍希釈
（６）泳動条件
１８ｍＡ定電流 ９０分間
First, Native PAGE electrophoresis was performed under the following conditions.
(1) Gel 4-20% Non-SDS PAGE mini 1mm 8well (manufactured by TEFCO)
(2) Electrophoretic layer Safety cell mini STC-808 (manufactured by TEFCO)
(3) Migration buffer 0.025M Trishydroxymethylaminomethane + 0.2M glycerin (4)
次に、以下の条件でウエスタンブロッティングを行った。
（１）ＰＶＤＦメンブレン
Ａｍｅｒｓｈａｍ Ｈｙｂｏｎｄ−Ｐ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）
（２）ブロッティングバッファー
２０％メタノール ＋ ２５ｍＭトリスヒドロキシメチルアミノメタン ＋ １９２ｍＭグリシン
（３）ブロッティング条件
６５ｍＡ定電流 ５０分間
（４）ブロッキングバッファー
２％ＥＣＬ Ｐｒｉｍｅ Ｂｌｏｃｋｉｎｇ Ｒｅａｇｅｎｔ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）＋ ０．１％Ｔｗｅｅｎ ２０ ＋ リン酸塩緩衝液（ＰＢＳ）
（５）ブロッキング条件
室温で１時間振盪後、冷蔵1晩
（６）一次抗体反応
Ａｎｔｉ−Ｈｕｍａｎ Ｍａｃ−２ｂｐ（８Ａ２）Ｍｏｕｓｅ ＩｇＧ（ＩＢＬ社製）
（７）二次抗体反応
ＨＲＰ． Ａｎｔｉ−Ｍｏｕｓｅ ＩｇＡ＋ＩｇＧ＋ＩｇＭ（Ｈ＋Ｌ）（ＫＰＬ社製）
（８）検出試薬
ＥＣＬ Ｐｒｉｍｅ Ｓｏｌｕｔｉｏｎ１ ＋ Ｓｏｌｕｔｉｏｎ２（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）
（９）検出機器
ＬＡＳ−１０００（富士フィルム社製）
Next, Western blotting was performed under the following conditions.
(1) PVDF Membrane Amersham Hybrid-P (manufactured by GE Healthcare)
(2)
(5) Blocking conditions After shaking at room temperature for 1 hour, refrigerate overnight (6) Primary antibody reaction Anti-Human Mac-2bp (8A2) Mouse IgG (manufactured by IBL)
(7) Secondary antibody reaction HRP. Anti-Mouse IgA + IgG + IgM (H + L) (manufactured by KPL)
(8) Detection reagent ECL Prime Solution1 + Solution2 (manufactured by GE Healthcare)
(9) Detection device LAS-1000 (manufactured by Fuji Film Co., Ltd.)
化学発光検出した画像を図３に示した。最上部のバンドを分子量９００ｋ以上のＭａｃ−２ｂｐとした。画像解析ソフト（ＣＳＡｎａｌｙｚｅｒ４，ＡＴＴＯ社製）を用いて、スタンダードを１として総Ｍａｃ−２ｂｐの濃度と分子量９００ｋ以上のＭａｃ−２ｂｐの濃度を計算した。総Ｍａｃ−２ｂｐの濃度の結果を比較例１（図４ａ）に分子量９００ｋ以上のＭａｃ−２ｂｐの濃度の結果を実施例１（図４ｂ）に示した。又、３枚のゲルを用いて、健常人の血清９例、ＮＡＳＨ患者の血清９例の総Ｍａｃ−２ｂｐの濃度と分子量９００ｋ以上のＭａｃ−２ｂｐの濃度の結果を図５に示した。 The image in which chemiluminescence was detected is shown in FIG. The uppermost band was Mac-2bp having a molecular weight of 900 k or more. Using image analysis software (CSAnalizer4, manufactured by ATTO), the concentration of total Mac-2bp and the concentration of Mac-2bp having a molecular weight of 900 k or more were calculated with the standard as 1. The result of the total concentration of Mac-2bp is shown in Comparative Example 1 (FIG. 4a), and the result of the concentration of Mac-2bp having a molecular weight of 900 k or more is shown in Example 1 (FIG. 4b). In addition, the results of the total Mac-2bp concentration and the Mac-2bp concentration of 900 k or more in molecular weight of 9 healthy subjects and 9 NASH patients using 3 gels are shown in FIG.
比較例１（図４ａ）のように総Ｍａｃ−２ｂｐ濃度では、健常人１例で１０を上回り、ＮＡＳＨ患者と同程度の値となったが、実施例１（図４ｂ）のように分子量９００ｋ以上のＭａｃ−２ｂｐ濃度で比べると健常人とＮＡＳＨ患者が明確に分かれた。検体数をそれぞれ９例に増やして測定した結果、図５のように平均値においては、総Ｍａｃ−２ｂｐ濃度（比較例）で健常人が７．０７に対してＮＡＳＨ患者が１７．０５と有意な増加が認められた。しかし、ＮＡＳＨ患者の低値の検体（７．７６）よりも高い値を示した健常人の検体が３検体（８．５５、１１．４６、１４．０７）存在した。この結果より、健常人とＮＡＳＨ患者で値の重なりが認められた。一方で、分子量９００ｋ以上のＭａｃ−２ｂｐ濃度（実施例）の平均値では健常人が２．０９に対してＮＡＳＨ患者が１０．９６と有意な増加が認められた。又、ＮＡＳＨ患者の低値の検体（４．３１）よりも健常人の高値の検体（２．９９）が低い値を示しており、健常人とＮＡＳＨ患者が明確に分かれた。以上より、実施例１は、比較例１、参考例１及び２に比べて、生体試料中の分子量９００ｋ以上のＭａｃ−２ｂｐ濃度により、健常人とＮＡＳＨ患者を明確に分けられることが明らかであった。 As shown in Comparative Example 1 (FIG. 4a), the total Mac-2bp concentration exceeded 10 in one healthy subject, which was about the same as that of NASH patients, but the molecular weight was 900 k as in Example 1 (FIG. 4b). When compared with the above Mac-2bp concentrations, healthy subjects and NASH patients were clearly separated. As a result of measuring by increasing the number of samples to 9 cases each, the average value as shown in FIG. 5 was significant with a total Mac-2bp concentration (comparative example) of 7.07 for healthy subjects and 17.05 for NASH patients. A significant increase was observed. However, there were 3 samples (8.55, 11.46, 14.07) of healthy subjects that showed higher values than the low values of NASH patients (7.76). From this result, overlapping values were observed in healthy subjects and NASH patients. On the other hand, the average value of the Mac-2bp concentration (Example) having a molecular weight of 900 k or more was 2.09 for healthy subjects and 10.96 for NASH patients, showing a significant increase. In addition, the high-value sample (2.99) of the healthy subject showed a lower value than the low-value sample (4.31) of the NASH patient, and the healthy subject and the NASH patient were clearly separated. From the above, it is clear that in Example 1, healthy subjects and NASH patients can be clearly separated by the Mac-2bp concentration in the biological sample having a molecular weight of 900 k or more, as compared with Comparative Example 1, Reference Examples 1 and 2. It was.
[実施例２]多量体のＭａｃ−２ｂｐの比較
分子量の比較を行うために、ＩＢＬ社製のＭａｃ−２ｂｐとＲ＆Ｄ ＳＹＳＴＥＭＳ社製のＲｅｃｏｍｂｉｎａｎｔ Ｈｕｍａｎ Ｇａｌｅｃｔｉｎ−３ＢＰ／ＭＡＣ−２ＢＰを実施例１と同様の方法でＮａｔｉｖｅ ＰＡＧＥ電気泳動及びウエスタンブロッティング法で評価した例を以下に示す。
[Example 2] Comparison of multimers Mac-2bp In order to compare molecular weights, Mac-2bp manufactured by IBL and Recombinant Human Galectin-3BP / MAC-2BP manufactured by R & D SYSTEMS were used in the same manner as in Example 1. An example evaluated by Native PAGE electrophoresis and Western blotting by the method is shown below.
化学発光検出した画像を図６に示した。Ｒ＆Ｄ ＳＹＳＴＥＭＳ社製のＭａｃ−２ｂｐは主に１０量体（約９００ｋ）又は１２量体（約１０８０ｋ）を形成しており、重量平均分子量が９５０ｋＤａである。ＩＢＬ社製のＭａｃ−２ｂｐのバンドも同じ位置で検出されているため、分子量９００ｋ以上又は１０量体以上のＭａｃ−２ｂｐを測定することにより、ＮＡＳＨやＮＡＦＬＤの進展度をより明確に判定することができる。 The image in which chemiluminescence was detected is shown in FIG. Mac-2bp manufactured by R & D SYSTEMS mainly forms a 10-mer (about 900 k) or a 12-mer (about 1080 k), and has a weight average molecular weight of 950 kDa. Since the band of Mac-2bp manufactured by IBL is also detected at the same position, the degree of progress of NASH and NAFLD can be more clearly determined by measuring Mac-2bp having a molecular weight of 900 k or more or a dimer or more. Can be done.
［実施例３及び比較例２］ラテックス凝集比濁法によるＮＡＳＨ患者及び健常人の血清Ｍａｃ−２ｂｐ値の比較
実施例３−（ａ）はＡｎｔｉ−Ｈｕｍａｎ Ｍａｃ−２ｂｐ（８Ａ２）Ｍｏｕｓｅ ＩｇＧを、実施例３−（ｂ）はＡｎｔｉ−Ｈｕｍａｎ Ｍａｃ−２ｂｐ（１２Ａ１）Ｍｏｕｓｅ ＩｇＧを、実施例３−（ｃ）はＡｎｔｉ−Ｈｕｍａｎ Ｍａｃ−２ｂｐ（６７Ａ１）Ｍｏｕｓｅ ＩｇＧを、実施例３−（ｄ）はＡｎｔｉ−Ｈｕｍａｎ Ｍａｃ−２ｂｐ（３１Ａ１）Ｍｏｕｓｅ ＩｇＧを使用してラテックス凝集試薬を調製して、ＮＡＳＨ患者及び健常人の血清中のＭａｃ−２ｂｐ濃度を測定した結果を示す（図７ａ、７ｂ、７ｃ、７ｄ）。なお、用いた４種の抗Ｍａｃ−２ｂｐ抗体はＩＢＬ社製である。
[Example 3 and Comparative Example 2] Comparison of serum Mac-2bp values of NASH patients and healthy subjects by the latex agglutination turbidimetric method Example 3- (a) was carried out using Anti-Human Mac-2bp (8A2) Mouse IgG. Example 3- (b) is Anti-Human Mac-2bp (12A1) Mouse IgG, Example 3- (c) is Anti-Human Mac-2bp (67A1) Mouse IgG, and Example 3- (d) is Anti. -Human Mac-2bp (31A1) Mouse IgG was used to prepare a latex agglutinating reagent, and the results of measuring the Mac-2bp concentration in the sera of NASH patients and healthy subjects are shown (FIGS. 7a, 7b, 7c, 7d). ). The four types of anti-Mac-2bp antibodies used are manufactured by IBL.
ラテックス凝集試薬は以下の様に調製した。
平均粒子径０．３μｍのポリスチレンラテックス（固形分１０％（Ｗ／Ｖ））１００μＬに、感作用緩衝液８００μＬを添加し、さらに抗Ｍａｃ−２ｂｐ抗体 １ｍｇ／ｍＬを１００μＬ添加した。従って感作時には１％（Ｗ／Ｖ）ラテックス液となる。この溶液を２５℃にて１時間振盪器にて１１０ｒｐｍで反応させた。その後、ブロッキング緩衝液を１ｍＬ添加し、２５℃にて１．５時間振盪器にて反応させた。その後、５℃、１５０００ｒｐｍにて３０分間遠心分離した。上清をアスピレータにて除去し、沈渣を得た。その沈渣にラテックス試薬用緩衝液を２ｍＬ加え、ボルテックスによりラテックスを分散させた。分散させたラテックス試薬を再び５℃、１５０００ｒｐｍにて３０分間遠心分離することにより沈渣を洗浄した。先ほどと同様に上清をアスピレータにて除去し、沈渣を得た。この沈渣にラテックス試薬用緩衝液を２ｍＬ加え、ボルテックスにて分散させた後、さらに３ｍＬのラテックス試薬用緩衝液を加えてよく攪拌後に超音波洗浄機にて分散処理を行い、固形分０．２％（Ｗ／Ｖ）のラテックス試薬とした。このようにして調整したラテックス試薬は４℃にて保存した。
The latex agglutination reagent was prepared as follows.
To 100 μL of polystyrene latex (
測定に使用した機器と測定条件は以下の通りである。
ラテックス試薬によるヒトＭａｃ−２ｂｐ量の測定は生化学用自動分析装置ビオリス２４ｉ Ｐｒｅｍｉｕｍ（東京貿易機械株式会社）を用いて行った。上記（１）で得られた固形分０．２％（Ｗ／Ｖ）のラテックス試薬をそのままＲ２試薬とした。測定条件は以下の通りである。
検体容量 １０μＬ
検体希釈液（Ｒ１試薬） ２００μＬ
試薬（Ｒ２試薬） ４０μＬ
測定波長 ７５０ｎｍ
測定温度 ３７℃
The equipment and measurement conditions used for the measurement are as follows.
The amount of human Mac-2bp using the latex reagent was measured using the biochemical automatic analyzer Biolis 24i Premium (Tokyo Boeki Holdings Corporation). The latex reagent having a solid content of 0.2% (W / V) obtained in (1) above was used as it was as an R2 reagent. The measurement conditions are as follows.
Specimen diluent (R1 reagent) 200 μL
Reagent (R2 reagent) 40 μL
Measurement wavelength 750 nm
Measurement temperature 37 ° C
Ｒ１試薬 ２００μＬを添加し引き続いて検体１０μＬを添加後、３７℃のセル内で５分間反応させる。その後、Ｒ２試薬 ４０μＬを添加し約３０秒後の吸光度と約３６０秒後の吸光度の差（△ＯＤ７５０）を測定し、吸光度変化量とした。また、検体の代わりに既知濃度の標準品をもちいて同様の測定を行い、予め検量線を作成しておき、上記検体の吸光度変化量を上記検量線に外挿して、検体中のヒトＭａｃ-２ｂｐ量を測定した。以上より、実施例３は、比較例２に比べて、健常人とＮＡＳＨ患者を明確に分けられることが明らかであった。 After adding 200 μL of R1 reagent and subsequently adding 10 μL of sample, the reaction is carried out in a cell at 37 ° C. for 5 minutes. Then, 40 μL of the R2 reagent was added, and the difference between the absorbance after about 30 seconds and the absorbance after about 360 seconds (ΔOD750) was measured and used as the amount of change in absorbance. Further, the same measurement is performed using a standard product having a known concentration instead of the sample, a calibration curve is prepared in advance, and the amount of change in the absorbance of the sample is extrapolated to the calibration curve to obtain human Mac- in the sample. The amount of 2 bp was measured. From the above, it was clear that in Example 3, healthy subjects and NASH patients could be clearly separated as compared with Comparative Example 2.
［実施例４］ラテックス凝集比濁法による炎症疾患、癌及び健常人の血清Ｍａｃ−２ｂｐ値の比較
実施例３−（ｂ）で使用した抗体を含むラテックス凝集試薬を用いて、炎症疾患（肝炎、肝硬変）、癌（肝臓癌、前立腺癌）及び健常人の血清中のＭａｃ−２ｂｐ濃度を測定した結果を示す（図８）。
[Example 4] Comparison of serum Mac-2bp values of inflammatory diseases, cancer and healthy subjects by the latex agglutination turbidimetric method Inflammatory diseases (hepatitis) using the latex agglutination reagent containing the antibody used in Example 3- (b). , Liver cirrhosis), cancer (liver cancer, prostate cancer) and the results of measuring the Mac-2bp concentration in the serum of healthy subjects are shown (FIG. 8).
Claims (7)
A test method for determining the possibility of suffering from hepatitis, liver cirrhosis, liver cancer, or prostate cancer, which comprises detecting a Mac-2bp concentration detected as having a molecular weight of 900 kDa or more in a blood sample.
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