JP4399593B2 - Influenza encephalopathy test method, marker consisting of protein expressed in human cerebrospinal fluid, diagnostic agent, diagnostic kit - Google Patents
Influenza encephalopathy test method, marker consisting of protein expressed in human cerebrospinal fluid, diagnostic agent, diagnostic kit Download PDFInfo
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Description
本発明は、インフルエンザ脳症の検査法、及び、ヒト髄液中に発現する１４−３−３タンパク質からなるマーカ、診断薬、診断キット関する。 The present invention relates to a test method for influenza encephalopathy, and a marker, diagnostic agent, and diagnostic kit comprising a 14-3-3 protein expressed in human cerebrospinal fluid.
ヒト髄液中にはアルブミンをはじめとして様々なタンパクが含有されている。髄液は脳表面および脳内部を循環することから、その成分はヒトの中枢神経疾患の病態を鋭敏に反映することが知られている。すなわち、脳出血では髄液中に赤血球が、髄膜炎や脳炎では白血球が、また多発性硬化症では免疫グロブリンが増加する。 Human cerebrospinal fluid contains various proteins including albumin. Since cerebrospinal fluid circulates on and within the brain, its components are known to reflect the pathology of human central nervous disease. That is, red blood cells increase in cerebrospinal fluid in cerebral hemorrhage, white blood cells increase in meningitis and encephalitis, and immunoglobulin increase in multiple sclerosis.
１４−３−３タンパク質は種を超えて高度に保存された２量体タンパク質であり、細胞の増殖分化、恒常性の維持、抗アポトーシス作用などを担っている。人体組織細胞中に広く存在するが、中でも脳神経細胞には細胞のタンパク重量の１％を占める程、豊富に含まれていることが知られている。 The 14-3-3 protein is a dimeric protein that is highly conserved across species, and is responsible for cell proliferation and differentiation, maintenance of homeostasis, anti-apoptotic action, and the like. Although widely present in human tissue cells, it is known that cerebral nerve cells are rich in occupying 1% of the protein weight of the cells.
１４−３−３タンパク質は正常髄液では検出されないが、異常プリオンタンパクによって発症するＣｒｅｕｚｆｅｌｄｔ−Ｊａｋｏｂ（クロイツフェルトヤコブ）病（以下、ＣＪＤ）の髄液において高率に検出されることから診断的価値が高いとされ、ＷＨＯ（世界保健機関）が定めるＣＪＤの診断基準にも１４−３−３タンパク質の検出が含まれるようになった。 14-3-3 protein is not detected in normal cerebrospinal fluid, but has a high diagnostic value because it is detected at a high rate in cerebrospinal fluid of Creuzfeldt-Jakob disease (hereinafter referred to as CJD) caused by abnormal prion protein The CJD diagnostic criteria set by the WHO (World Health Organization) now include the detection of 14-3-3 protein.
髄液中１４−３−３タンパク質は、ＣＪＤ以外にも脳腫瘍、脳出血、脳梗塞、血管性痴呆、髄膜炎、脊髄炎でも検出されることが報告されており、脳組織崩壊を反映する可能性が示唆されている。これらの報告はすべて成人領域であって、小児領域（おもに１８歳以下）での集約された報告は現在までない。 14-3-3 protein in cerebrospinal fluid has been reported to be detected in brain tumor, cerebral hemorrhage, cerebral infarction, vascular dementia, meningitis and myelitis in addition to CJD, and may reflect brain tissue disruption Sex has been suggested. These reports are all in the adult area, and there are no aggregate reports to date in the pediatric area (mainly under 18 years).
１４−３−３タンパク質はそのアミノ酸配列の差異から７つのアイソフォーム、β、γ、ζ、θ、ε、σ、ηが知られている。それぞれのアイソフォームはアポトーシスシグナル、増殖分化、細胞内輸送、細胞周期調節などの異なる機能を持つとされるが詳細は不明である。 The 14-3-3 protein is known to have seven isoforms, β, γ, ζ, θ, ε, σ, and η, due to differences in amino acid sequences. Each isoform has different functions such as apoptotic signal, proliferation differentiation, intracellular transport, cell cycle regulation, etc., but details are unknown.
１４−３−３タンパク質の７つのアイソフォームの脳内における分布は、マウスの研究からβ、ζ、γ、ηは神経細胞体と神経核に、εは中枢神経灰白質に、ηは小脳核、視床下部核、神経細胞体、神経核に含まれることが報告されている。 The distribution of seven isoforms of 14-3-3 protein in the brain was determined from mouse studies, β, ζ, γ, and η are in the neuronal cell body and nucleus, ε is in the central nerve gray matter, and η is in the cerebellar nucleus. It has been reported to be contained in the hypothalamic nucleus, nerve cell body, and nerve nucleus.
髄液中に検出される１４−３−３タンパク質のアイソフォームは疾患によって特異性があることが知られている。例えばＣＪＤの髄液ではアイソフォームのうちβ、γ、η、εが検出され、またアルツハイマー病の髄液では常にηが検出されることが報告されているが、そのメカニズムについては不明であり、これらアイソフォームのパターン分析が臨床診断に結びつく可能性も示唆されている。 It is known that the isoform of 14-3-3 protein detected in cerebrospinal fluid has specificity depending on the disease. For example, CJD cerebrospinal fluid has been reported to detect β, γ, η, ε among isoforms, and CSF is always detected in cerebrospinal fluid of Alzheimer's disease, but the mechanism is unknown, It is suggested that pattern analysis of these isoforms may lead to clinical diagnosis.
インフルエンザ脳症は、インフルエンザウイルス感染に伴っておきる急性脳症であって、インフルエンザ脳炎を含み、小児に頻発し予後不良の疾患である。脳組織および髄液中にウイルスゲノムがほとんど認められず、また治療法も確立されていないことから、小児難病の一つとされてきた。 Influenza encephalopathy is an acute encephalopathy associated with influenza virus infection, including influenza encephalitis, a frequent disease in children and a poor prognosis. It has been regarded as one of the intractable diseases of children because almost no viral genome is found in brain tissue and cerebrospinal fluid, and no cure has been established.
小児神経疾患には、難治性神経疾患が数多くあるが、中でもインフルエンザ脳症はインフルエンザ感染に伴い小児にけいれんや意識障害が生じ、急性増悪して死の転帰を迎える予後不良疾患である。インフルエンザ感染時には高熱を来たすため、けいれん意識障害の初期には、良性である熱性けいれんか、あるいは予後不良のインフルエンザ脳症か臨床的に区別することが困難である。この初期の段階で明らかにインフルエンザ脳症であることを診断できれば、きわめて早期から集中治療を行い臨床的予後が改善できる可能性が考えられる。しかしながら現在まで初期にインフルエンザ脳症であることを診断できる方法は存在せず、早期診断のためのマーカーの開発が期待されている。 There are many intractable neurological diseases in childhood, but influenza encephalopathy is a disease with a poor prognosis that causes convulsions and impaired consciousness in children due to influenza infection, resulting in acute exacerbations and death. It is difficult to clinically distinguish benign febrile convulsions or influenza encephalopathy with a poor prognosis in the early stages of convulsive consciousness because high fever occurs during influenza infection. If it can be clearly diagnosed as influenza encephalopathy at this early stage, it may be possible to improve clinical prognosis by intensive treatment from an extremely early stage. However, there is no method capable of diagnosing influenza encephalopathy at an early stage until now, and development of a marker for early diagnosis is expected.
また急性期の脳障害の指標となる髄液タンパクは、ＮＳＥ（Ｎｅｕｒｏｎ Ｓｐｅｃｉｆｉｃ Ｅｎｏｌａｓｅ：神経特異性エノラーゼ）、ＭＢＰ（Ｍｙｅｌｉｎ Ｂａｓｉｃ Ｐｒｏｔｅｉｎ）が知られているが、必ずしも病態を鋭敏に反映するとは言えず、適切な病態把握および予後判定のための信頼性の高い髄液タンパクの確立が急務とされている。
本発明は、こうした事情に鑑みて、とくに小児神経疾患の早期診断のための検査法、マーカ、診断薬、診断キットを開発することを目的とするものである。 In view of such circumstances, an object of the present invention is to develop a test method, a marker, a diagnostic agent, and a diagnostic kit for early diagnosis of childhood neurological diseases.
本発明は、発明者のヒト中枢神経疾患における髄液解析に関しての研究の中で、１４−３−３タンパク質の髄液中での検出が疾患診断のみならず病態解析に有用であることを解明した結果に基づくものであり、（１）ヒト髄液中に含まれる１４−３−３タンパク質を指標としたインフルエンザ脳症の検査法、（２）１４−３−３タンパク質のアイソフォームにおいて、β、θ、γ、ζ、εの順に強く検出された場合に、インフルエンザ脳症であると判断する前記記載の検査方法、（３）検査対象が、小児インフルエンザ脳症であり、ヒト髄液が小児髄液である前記記載検査方法、（４）インフルエンザ脳症の診断に用いるタンパク質マーカであって、ヒト髄液中に発現する１４−３−３タンパク質からなるマーカ、（５）１４−３−３−タンパク質が、β、θ、γ、ζおよびεのアイソフォームである前記記載のマーカ、（６）診断対象が、小児インフルエンザ脳症であり、ヒト髄液が小児髄液である前記記載のマーカ、（７）インフルエンザ脳症の診断薬であって、ヒト髄液中に発現する１４−３−３タンパク質に特異的に結合する抗体を含む診断薬、（８）前記抗体が、１４−３−３タンパク質におけるβ、θ、γ、ζおよびεのアイソフォームのそれぞれに特異的に結合する抗体を含む診断薬、（９）１４−３−３タンパク質が、小児髄液中に発現するタンパク質である前記記載の診断薬、（１０）前記抗体がモノクローナル抗体である前記記載の診断薬、（１１）インフルエンザ脳症の診断キットであって、ヒト髄液中に発現する１４−３−３タンパク質に特異的に結合する一次抗体と、この一次抗体に特異的に結合し、かつ識別標識を有する二次抗体とを含み、前記識別標識の検出により前記１４−３−３タンパク質を検出する診断キット、（１２）前記一次抗体が、１４−３−３タンパク質におけるβ、θ、γ、ζおよびεのアイソフォームのそれぞれに特異的に結合する抗体を含む前記記載の診断キット、（１３）１４−３−３タンパク質が、小児髄液中に発現するタンパク質である前記記載の診断キット、（１４）前記一次抗体が、ウサギ由来の抗体であり、前記二次抗体がヤギ由来の抗体である前記記載の診断キット、（１５）前記一次抗体および二次抗体が、それぞれモノクローナル抗体である前記記載の診断キット、（１６）前記識別標識が、酵素標識若しくは蛍光標識である前記記載の診断キット、に関する。 The present invention elucidates that detection of 14-3-3 protein in cerebrospinal fluid is useful not only for disease diagnosis but also for pathological analysis in the inventor's research on cerebrospinal fluid in human central nervous disease (1) a test method for influenza encephalopathy using 14-3-3 protein as an index contained in human cerebrospinal fluid, (2) in the isoform of 14-3-3 protein, (3) The test method according to the above, wherein it is determined that the encephalopathy is influenza encephalopathy when strongly detected in the order of θ, γ, ζ, ε; (3) the test object is pediatric influenza encephalopathy; (4) A protein marker used for diagnosis of influenza encephalopathy, the marker comprising 14-3-3 protein expressed in human cerebrospinal fluid, (5) 14-3-3 protein The marker according to the above, wherein the quality is an isoform of β, θ, γ, ζ, and ε, (6) the marker according to the above, wherein the diagnosis target is pediatric influenza encephalopathy, and the human cerebrospinal fluid is pediatric cerebrospinal fluid, 7) a diagnostic agent for influenza encephalopathy, which comprises an antibody that specifically binds to 14-3-3 protein expressed in human cerebrospinal fluid, and (8) said antibody in 14-3-3 protein The diagnostic agent comprising an antibody that specifically binds to each of the β, θ, γ, ζ, and ε isoforms, (9) wherein the 14-3-3 protein is a protein expressed in pediatric cerebrospinal fluid (10) The diagnostic agent according to the above, wherein the antibody is a monoclonal antibody, (11) a diagnostic kit for influenza encephalopathy, which specifically binds to 14-3-3 protein expressed in human spinal fluid Primary antibody A diagnostic kit that specifically binds to the primary antibody and has a discrimination label, and detects the 14-3-3 protein by detecting the discrimination label, (12) the primary antibody comprises: The diagnostic kit as described above, comprising an antibody that specifically binds to each of β, θ, γ, ζ, and ε isoforms in 14-3-3 protein, (13) 14-3-3 protein is pediatric spinal fluid (14) The diagnostic kit according to the above, wherein the primary antibody is a rabbit-derived antibody, and the secondary antibody is a goat-derived antibody, (15) the primary kit The diagnostic kit according to the above, wherein the antibody and the secondary antibody are each a monoclonal antibody, and (16) the diagnostic kit according to the above, wherein the identification label is an enzyme label or a fluorescent label.
本発明によると、インフルエンザ感染症初期に伴うけいれん、意識障害の症状の中から、インフルエンザ脳症を高率に診断することができ、集中治療に結びつけることが可能である。インフルエンザ脳症は死亡率も高く、本発明が臨床現場における同疾患を早期に鑑別することにより、救命率の向上および神経学的予後の改善に結びつくことが予想される。また１４−３−３アイソフォームを分析することにより、疾患の診断ならびに脳障害部位の推定が可能になる。 According to the present invention, it is possible to diagnose influenza encephalopathy at a high rate from the symptoms of convulsions and impaired consciousness associated with the early stages of influenza infection, and it is possible to link to intensive care. Influenza encephalopathy also has a high mortality rate, and the present invention is expected to lead to an improvement in the survival rate and an improvement in neurological prognosis by early differentiation of the disease in the clinical field. Further, by analyzing the 14-3-3 isoform, it becomes possible to diagnose a disease and estimate a brain damage site.
以下、この発明の実施形態を具体的に説明する。 Embodiments of the present invention will be specifically described below.
本発明において、検査対象またはマーカとなるのは、ヒト髄液中に含まれる１４−３−３タンパク質である。このタンパク質は、７つのアイソフォームを持つことが知られているが、インフルエンザ脳症においてヒト髄液中に発現するは、β、θ、γ、ζ、εであり、かつこの順に強く検出される。したがって、このアイソフォームの発現パターンの検査により、より正確にインフルエンザ脳症であるか否かを判断できる。さらに、アイソフォームの発現パターン分析により、脳のどの部分に障害が生じているかを予測することができ、この結果、脳症に対する適切な処置をとることができる。 In the present invention, 14-3-3 protein contained in human cerebrospinal fluid serves as a test object or marker. Although this protein is known to have seven isoforms, β, θ, γ, ζ, and ε are expressed in human cerebrospinal fluid in influenza encephalopathy, and are strongly detected in this order. Therefore, by examining the expression pattern of this isoform, it can be more accurately determined whether or not the patient has influenza encephalopathy. Furthermore, by analyzing the expression pattern of isoforms, it is possible to predict which part of the brain is damaged, and as a result, an appropriate treatment for encephalopathy can be taken.
１４−３−３タンパク質の７つのアイソフォームのアミノ酸配列はすでに同定されておりその相違を示す（図５）。その中でもセリン、スレオニン残基はリン酸化を受ける部位であり、アイソフォーム間で相同性が高く、またリン酸化を受けることにより１４−３−３タンパク質自身の機能変化が予想されるため、同部位を特に太線で強調した。 The amino acid sequences of the seven isoforms of 14-3-3 protein have already been identified and show the differences (FIG. 5). Among them, serine and threonine residues are sites that undergo phosphorylation, and are highly homologous between isoforms, and functional changes of the 14-3-3 protein itself are expected due to phosphorylation. Was highlighted with a bold line.
前記１４−３−３タンパク質の検出方法は、特に制限されないが、抗体を用いた検出方法が好ましい。前記抗体は、特に制限されないが、例えば、ウサギ、ヤギ、マウス、ラット、ブタ、ヒツジ、イヌなどの各種の動物由来のものが使用できる。また、抗体の作製は、１４−３−３タンパク質を用い、一般の方法で、前記動物を免疫することにより実施できる。前記抗体としては、モノクローナル抗体が好ましい。 The method for detecting the 14-3-3 protein is not particularly limited, but a detection method using an antibody is preferable. The antibody is not particularly limited, and for example, antibodies derived from various animals such as rabbits, goats, mice, rats, pigs, sheep and dogs can be used. The antibody can be prepared by immunizing the animal by a general method using 14-3-3 protein. The antibody is preferably a monoclonal antibody.
前記抗体を用いた検出方法は、特に制限されないが、検出精度と操作方法の簡便性からＥＬＩＳＡ法が好ましい。ＥＬＩＳＡ法は、検出対象タンパク質（本発明では、１４−３−３タンパク質、若しくはそのβ、θ、γ、ζ、εのアイソフォーム）に特異的に結合する一次抗体と、この一次抗体に特異的に結合し、かつ酵素標識（ラベル）を有する二次抗体を使用する方法である。前記識別標識としては、後述のように酵素標識の他に、蛍光標識やＡＢＣ法を使用してもよい。ＥＬＩＳＡ法は、特に制限されず、後述の実施例のように、髄液をＳＤＳ−ＰＡＧＥで電気泳動したあと、ウエスタンブロッティングによりタンパク質をニトロセルロース膜に転写し、これに一次抗体および二次抗体を添加し、前記二次抗体の識別標識を検出することにより実施することができる。この他に、ポリスチレンビーズなどの固相に一次抗体を結合させ、これに髄液を供給して１４−３−３タンパク質を結合させ、この状態で、Ｂ／Ｆ分離を行い、そして、１４−３−３タンパク質に特異的に結合し、かつ標識を有する二次抗体を添加して前記タンパク質に結合させ、前記標識を検出するサンドイッチ法であってもよい。 The detection method using the antibody is not particularly limited, but the ELISA method is preferable from the viewpoint of detection accuracy and simplicity of operation method. The ELISA method comprises a primary antibody that specifically binds to a protein to be detected (in the present invention, a 14-3-3 protein, or its β, θ, γ, ζ, and ε isoforms), and is specific to this primary antibody. And a secondary antibody having an enzyme label (label). As the identification label, a fluorescent label or ABC method may be used in addition to the enzyme label as described later. The ELISA method is not particularly limited, and as shown in the examples below, the cerebrospinal fluid is electrophoresed by SDS-PAGE, then the protein is transferred to a nitrocellulose membrane by Western blotting, and the primary antibody and the secondary antibody are added thereto. It can be carried out by adding and detecting the identification label of the secondary antibody. In addition, the primary antibody is bound to a solid phase such as polystyrene beads, and cerebrospinal fluid is supplied to the primary antibody to bind the 14-3-3 protein. In this state, B / F separation is performed, and 14- It may be a sandwich method in which a secondary antibody that specifically binds to 3-3 protein and has a label is added to bind to the protein and the label is detected.
酵素標識の具体例として、Ｈｏａｒｓｅ Ｒａｄｄｉｓｈ Ｐｅｒｏｘｉｄａｓｅ（ＨＲＰ）を用いる方法がある。これにより蛍光発色させることが可能となり、１４−３−３タンパク質を可視化同定することが可能である。 As a specific example of enzyme labeling, there is a method using Hoarse Raddish Peroxidase (HRP). As a result, it is possible to develop a fluorescent color and to visualize and identify the 14-3-3 protein.
なお、本発明における「β、θ、γ、ζおよびεのアイソフォームのそれぞれに特異的に結合する抗体」とは、それぞれのアイソフォームの特異的アミノ酸配列を認識して結合する免疫グロブリン抗体を、
同じく本発明における「モノクロナール抗体」とは、単一免疫Ｂ細胞より分泌される単一の免疫グロブリンからなる抗体群を、
同じく本発明における「ウサギ由来の抗体」とは、ウサギに１４−３−３タンパク質（アイソフォーム）を免疫接種することにより血清中から回収される免疫グロブリン抗体を、
同じく本発明における「ヤギ由来の抗体」とは、ヤギに１４−３−３タンパク質（アイソフォーム）を免疫接種することにより血清中から回収される免疫グロブリン抗体を、
同じく本発明における「酵素標識」とは、二次抗体の標識にＨＲＰ（Ｈｏｒｓｅｒａｄｉｓｈ ｐｅｒｏｘｉｄａｓｅ，西洋わさび過酸化酵素）やＡＰ（ａｌｋａｌｉｎｅ ｐｈｏａｐｈｔａｓｅ、アルカリフォスファターゼ）などの酵素を用いて、酵素反応の結果生じた沈殿物の色素を検出する方法を、
同じく本発明における「蛍光標識」とは、二次抗体の標識にＦＩＴＣ（ｆｌｕｏｒｅｓｃｅｉｎ ｉｓｏｔｈｉｏｃｙａｎａｔｅ：緑色蛍光を発する）やローダミン（ｒｈｏｄａｍｉｎｅ：赤色蛍光を発する）などを用いて、それぞれに対応する波長の励起光を当てて蛍光を発検出する方法を、
同じく本発明における「ＡＢＣ法」とは、二次抗体の標識にビオチン（ｂｉｏｔｉｎ）を用いるが、このビオチンにアビジンと酵素の複合体を結合させることで、抗体１分子あたりに結合する酵素の量が増し、高感度に検出できる方法を、
いう。In the present invention, “an antibody that specifically binds to each of the isoforms of β, θ, γ, ζ, and ε” refers to an immunoglobulin antibody that recognizes and binds to a specific amino acid sequence of each isoform. ,
Similarly, “monoclonal antibody” in the present invention refers to an antibody group consisting of a single immunoglobulin secreted from a single immune B cell,
Similarly, the “rabbit-derived antibody” in the present invention refers to an immunoglobulin antibody recovered from serum by immunizing a rabbit with 14-3-3 protein (isoform),
Similarly, the “goat-derived antibody” in the present invention refers to an immunoglobulin antibody recovered from serum by immunizing a goat with 14-3-3 protein (isoform),
Similarly, the term “enzyme labeling” in the present invention is the result of an enzyme reaction using an enzyme such as HRP (horseradish peroxidase) or AP (alkaline phosphatase) for labeling a secondary antibody. How to detect the pigment in the precipitate
Similarly, the “fluorescent label” in the present invention uses FITC (fluorescein isothiocyanate: emits green fluorescence), rhodamine (rhodamine: emits red fluorescence), etc. for the labeling of the secondary antibody. How to detect fluorescence by applying
Similarly, the “ABC method” in the present invention uses biotin for labeling the secondary antibody. By binding a complex of avidin and enzyme to this biotin, the amount of enzyme bound per antibody molecule The method that can be detected with high sensitivity,
Say.
ヒト髄液中の１４−３−３タンパクを検出するために、まず採取された髄液を遠心分離し（３０００ｇ、１０分間）、髄液中に含まれる細胞成分を除去した。上清を採取しＭｉｃｒｏｃｏｎ ＹＭ−１０（Ｍｉｌｌｉｐｏｒｅ社製、ＵＳＡ）を用いて遠心分離（１３０００ｇ）を行うことにより髄液を１０倍に濃縮させた。濃縮した髄液はＳＤＳサンプルバッファーを加えて９５度で５分間熱いた。その後髄液タンパク３０μｇ量を１２．５％ＳＤＳ−ＰＡＧＥで電気泳動し、ウエスタンブロッティング（１５Ｖ、１２０分）を行うことによりニトロセルロース膜に髄液タンパクを転写した。転写後５％ミルクＴＢＳ−Ｔ液に浸して、常温で１５分間振盪しニトロセルロース膜上のタンパクを固定した。１次抗体としてウサギ抗１４−３−３β抗体（ｓｃ−６２７，Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ社，ＵＳＡ）を１：１００濃度で４℃で一晩反応させ、さらに２次抗体としてＨＲＰ標識ヤギ抗ウサギＩｇＧ抗体（ｓｃ−２００４、Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ社，ＵＳＡ）を１：２５００濃度で常温にて１時間反応させた。１４−３−３タンパク質はＥＣＬキット（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅ社，ＵＳＡ）を用いてバンドとして可視化した。また、１４−３−３タンパク質が検出された場合には、そのアイソフォームを検出するためそれぞれの１４−３−３タンパク質のアイソフォーム特異抗体、ａｎｔｉ−β ａｎｔｉｂｏｄｉｅｓ（ｓｃ−６２８，Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，ＵＳＡ），ａｎｔｉ−γ ａｎｔｉｂｏｄｉｅｓ（ｓｃ−７３１，Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，ＵＳＡ），ａｎｔｉ−ζ ａｎｔｉｂｏｄｉｅｓ（ｓｃ−１０１９，Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，ＵＳＡ），ａｎｔｉ−ε ａｎｔｉｂｏｄｉｅｓ（ｓｃ−１０２０，Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，ＵＳＡ），ａｎｔｉ−η ａｎｔｉｂｏｄｉｅｓ（ｓｃ−１７２８６，Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，ＵＳＡ），ａｎｔｉ−θ ａｎｔｉｂｏｄｉｅｓ（ｓｃ−７３２，Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，ＵＳＡ），ａｎｄ ａｎｔｉ−σ ａｎｔｉｂｏｄｉｅｓ（ｓｃ−７６８１，Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，ＵＳＡ））、ないし、Ａｎｔｉ−Ｈｕｍａｎ １４−３−３ Ｐｒｏｔｅｉｎ Ｒａｂｂｉｔ ＩｇＧ Ａｆｆｉｎｉｔｙ Ｐｕｒｉｆｙ（１８６４９，ＩＢＬ，Ｊａｐａｎ），Ａｎｔｉ−Ｈｕｍａｎ １４−３−３ β Ｐｒｏｔｅｉｎ Ｒａｂｂｉｔ ＩｇＧ Ａｆｆｉｎｉｔｙ Ｐｕｒｉｆｙ（１８６４１，ＩＢＬ，Ｊａｐａｎ），Ａｎｔｉ−Ｈｕｍａｎ １４−３−３ γ Ｐｒｏｔｅｉｎ（６３Ａ１）Ｍｏｕｓｅ ＩｇＧ ＭｏＡｂ（１００３７，ＩＢＬ，Ｊａｐａｎ），Ａｎｔｉ−Ｈｕｍａｎ １４−３−３ γ Ｐｒｏｔｅｉｎ Ｒａｂｂｉｔ ＩｇＧ Ａｆｆｉｎｉｔｙ Ｐｕｒｉｆｙ（１８６４７，ＩＢＬ，Ｊａｐａｎ），Ａｎｔｉ−Ｈｕｍａｎ １４−３−３ ε Ｐｒｏｔｅｉｎ Ｒａｂｂｉｔ ＩｇＧ Ａｆｆｉｎｉｔｙ Ｐｕｒｉｆｙ（１８６４３，ＩＢＬ，Ｊａｐａｎ），Ａｎｔｉ−Ｈｕｍａｎ １４−３−３ ζ Ｐｒｏｔｅｉｎ Ｒａｂｂｉｔ ＩｇＧ Ａｆｆｉｎｉｔｙ Ｐｕｒｉｆｙ（１８６４４，ＩＢＬ，Ｊａｐａｎ），Ａｎｔｉ−Ｈｕｍａｎ１４−３−３ η Ｐｒｏｔｅｉｎ Ｒａｂｂｉｔ ＩｇＧ Ａｆｆｉｎｉｔｙ Ｐｕｒｉｆｙ，（１８６４５，ＩＢＬ，Ｊａｐａｎ），Ａｎｔｉ−Ｈｕｍａｎ １４−３−３ σ Ｐｒｏｔｅｉｎ（６９）Ｒａｂｂｉｔ ＩｇＧ Ａｆｆｉｎｉｔｙ Ｐｕｒｉｆｙ（１８６４０，ＩＢＬ，Ｊａｐａｎ），Ａｎｔｉ−Ｈｕｍａｎ １４−３−３ σ Ｐｒｏｔｅｉｎ（Ｃ）Ｒａｂｂｉｔ ＩｇＧ Ａｆｆｉｎｉｔｙ Ｐｕｒｉｆｙ（１８６４２，ＩＢＬ，Ｊａｐａｎ），Ａｎｔｉ−Ｈｕｍａｎ １４−３−３ σ Ｐｒｏｔｅｉｎ（Ｎ）Ｒａｂｂｉｔ ＩｇＧ Ａｆｆｉｎｉｔｙ Ｐｕｒｉｆｙ（１８６４６．ＩＢＬ，Ｊａｐａｎ），Ａｎｔｉ−Ｈｕｍａｎ １４−３−３ τ Ｐｒｏｔｅｉｎ（３３Ａ）Ｍｏｕｓｅ ＩｇＧ ＭｏＡｂ（１００１７，ＩＢＬ，Ｊａｐａｎ）を用いて、それぞれのアイソフォーム同定を行った。この一連の作業を、インフルエンザ感染症、インフルエンザ脳症、非インフルエンザ感染の急性脳症の３群のみならず、小児神経疾患全般についても検討した。 In order to detect 14-3-3 protein in human cerebrospinal fluid, the collected cerebrospinal fluid was first centrifuged (3000 g, 10 minutes) to remove cellular components contained in the cerebrospinal fluid. The supernatant was collected and centrifuged (13000 g) using Microcon YM-10 (Millipore, USA) to concentrate the
図１は、小児神経疾患の中で髄液中に１４−３−３タンパク質が検出された具体例を示す。この結果から、髄液中１４−３−３タンパク質は、インフルエンザ脳症（ＩＡＥ）、ミトコンドリア異常症（ＭＥＬＡＳ）、Ｈｅｍｉｃｏｎｖｕｌｓｉｏｎ−Ｈｅｍｉｐｌｅｇｉａ症候群（ＨＨ症候群）、ウイルス性および細菌性髄膜炎、脊髄小脳変性症、悪性リンパ腫で検出されることがわかった。 FIG. 1 shows a specific example in which 14-3-3 protein was detected in cerebrospinal fluid in pediatric neurological diseases. From this result, 14-3-3 protein in cerebrospinal fluid was found to be influenza encephalopathy (IAE), mitochondrial disorder (MELAS), Hemicconvection-Hemiplegia syndrome (HH syndrome), viral and bacterial meningitis, spinocerebellar degeneration Found to be detected in malignant lymphoma.
図２は髄液中に１４−３−３タンパク質が検出された例の頭部ＭＲＩ写真を示す。ＨＨ症候群、インフルエンザ脳症、脊髄小脳変性症でそれぞれ障害されている脳部位を矢印で示した。これらの結果から、矢印で示された患者の脳障害部位が髄液中の１４−３−３タンパク質検出の責任病巣であることがわかった。また、これらの障害部位と１４−３−３タンパク質アイソフォームが関連することも推測できる。 FIG. 2 shows a head MRI photograph of an example in which 14-3-3 protein was detected in the cerebrospinal fluid. The brain regions that are impaired due to HH syndrome, influenza encephalopathy, and spinocerebellar degeneration are indicated by arrows. From these results, it was found that the brain injury site of the patient indicated by the arrow is the responsible lesion for detecting 14-3-3 protein in the cerebrospinal fluid. It can also be assumed that these damaged sites are associated with 14-3-3 protein isoforms.
図３は、インフルエンザ感染症のみで脳症非発生例、インフルエンザ脳症例、非インフルエンザ感染症に伴う脳症例の臨床的特徴および髄液データである。この結果から、インフルエンザ脳症が小児で発生しやすいこと、一般に行われる髄液検査だけではインフルエンザ脳症かどうか区別できないこと、インフルエンザタイプＢでもインフルエンザ脳症を発症しうることが判明した。 FIG. 3 shows clinical characteristics and cerebrospinal fluid data of a case of non-encephalopathy with only influenza infection, a case of influenza brain, a brain case with non-influenza infection. From these results, it was found that influenza encephalopathy is likely to occur in children, that it is not possible to distinguish whether it is influenza encephalopathy only by a commonly performed cerebrospinal fluid test, and that influenza encephalopathy can also develop influenza encephalopathy.
図３は、インフルエンザ感染症のみで脳症非発生例、インフルエンザ脳症例、非インフルエンザ感染症に伴う脳症例における髄液中１４−３−３タンパク質の検出を示すウエスタンブロットの結果であり、この結果から、髄液中１４−３−３タンパク質はインフルエンザ脳症に特異的に検出され、インフルエンザ脳症の診断に有用であることがわかった。 FIG. 3 shows the results of Western blotting showing detection of 14-3-3 protein in cerebrospinal fluid in encephalopathy-free cases with influenza infection alone, influenza brain cases, and brain cases with non-influenza infections. The 14-3-3 protein in cerebrospinal fluid was detected specifically in influenza encephalopathy, and was found to be useful for diagnosis of influenza encephalopathy.
図４は、インフルエンザ脳症例およびミトコンドリア病（ＭＥＬＡＳ）例における１４−３−３タンパク質のアイソフォームの同定に成功したウエスタンブロットの結果であり、この結果から、１４−３−３タンパク質の７つのアイソフォームの中でβ、γ、ζ、θ、εの５つがインフルエンザ脳症で陽性であることがわかる。またこの５つのアイソフォームの中でもインフルエンザ脳症ではβ、θ、γ、ζ、εの順に強く検出されることがわかる。一方、ミトコンドリア病（ＭＥＬＡＳ）ではβ、ζの２つが陽性であり、ほぼ同程度に検出されることがわかった。 FIG. 4 shows the results of a Western blot that succeeded in identifying isoforms of 14-3-3 protein in influenza brain cases and mitochondrial disease (MELAS) cases. It can be seen that 5 of β, γ, ζ, θ, and ε are positive in influenza encephalopathy. It can also be seen that among these five isoforms, influenza encephalopathy is detected strongly in the order of β, θ, γ, ζ, and ε. On the other hand, in mitochondrial disease (MELAS), both β and ζ were positive and were found to be detected at almost the same level.
図６は、ミトコンドリア病（ＭＥＬＡＳ）患者における病期における頭部ＭＲＩ画像である。障害部位は矢印で示してある。この結果から、病期によって障害される脳部位が変化していることがわかる。 FIG. 6 is a head MRI image at a stage in a mitochondrial disease (MELAS) patient. The site of failure is indicated by an arrow. From this result, it can be seen that the brain region that is damaged by the stage changes.
図７は、ミトコンドリア病（ＭＥＬＡＳ）患者における臨床症状と髄液中１４−３−３タンパク質の検出変化を示したものである。ＭＲＩは図６で示されたものを病期に当てはめてある。この結果から、髄液中１４−３−３タンパク質は障害が進行するたびに髄液中で検出されること、中でも脳卒中発作時にもつとも強く検出されること、これらは頭部ＭＲＩで後頭葉と側頭葉が中心であること、これらは臨床上の急性期と一致して出現し安定すると消退することがわかった。 FIG. 7 shows clinical symptoms and detection changes of 14-3-3 protein in cerebrospinal fluid in patients with mitochondrial disease (MELAS). MRI is applied to the stage shown in FIG. From this result, it can be seen that the 14-3-3 protein in the cerebrospinal fluid is detected in the cerebrospinal fluid every time the disorder progresses, and is especially strongly detected at the time of stroke. It was found that the lobe is central, and that these appear in accord with the clinical acute phase and disappear when stable.
図８は、Ｂａｘｔｅｒらによって報告されたマウスの脳における１４−３−３タンパク質の脳内分布を示す。これらより１４−３−３タンパク質のアイソフォームが脳の一部に限局して存在しており、髄液中に検出される１４−３−３タンパク質のアイソフォームと一致してすることが予想できる。 FIG. 8 shows the brain distribution of 14-3-3 protein in the mouse brain reported by Baxter et al. From these, it can be expected that the isoform of the 14-3-3 protein is localized in a part of the brain and coincides with the isoform of the 14-3-3 protein detected in the cerebrospinal fluid. .
図９は、髄液中１４−３−３タンパク質の検出メカニズムである。これによると、髄液中１４−３−３タンパク質が検出されるメカニズムには、急性期の脳破壊に付随して検出されるものと、慢性期のアポトーシス刺激に随伴して髄液中に放出されるものの、２種類があることがわかる。前者にはインフルエンザ脳症などが、後者には脊髄小脳変性症などが疾患として該当する。 FIG. 9 shows the detection mechanism of 14-3-3 protein in cerebrospinal fluid. According to this, there are two mechanisms for detecting 14-3-3 protein in cerebrospinal fluid, one that is detected accompanying brain destruction in the acute phase, and one that is released into the cerebrospinal fluid in association with apoptotic stimulation in the chronic phase. However, there are two types. The former includes influenza encephalopathy, and the latter includes spinocerebellar degeneration.
本研究成果の要点は、以下のとおりである。
１．１４−３−３タンパク質はインフルエンザ脳症の患者髄液で全例に検出され、インフルエンザ感染症の高熱に伴う熱性けいれん等の意識障害では１例も検出されなかった。このことは、髄液中１４−３−３タンパク質の検出が、インフルエンザ脳症の早期診断に有用であることを明確に示している。
２．１４−３−３タンパク質は同様にインフルエンザ感染症以外に伴う急性脳症でも検出されず、急性脳症の中でもインフルエンザ脳症を明確に診断するために有用であった。
３．インフルエンザ脳症患者群の髄液からは、１４−３−３アイソフォームとしてβ、γ、ζ、θ、εが検出され、ヒトの脳が広範囲に障害されていることが明らかになった。
４．５つのアイソフォームの中でもβ、θ、γ、ζ、εの順に強く検出されることは、インフルエンザ脳症で特異的なアイソフォーム出現パターンであると考えられる。
５．これら５つの１４−３−３アイソフォームの出現パターンの同定は、障害された脳部位の同定が可能になり、また髄液中に１４−３−３タンパクが検出されるメカニズムの解明に有用である。
６．ミトコンドリア病（ＭＥＬＡＳ）では、髄液中１４−３−３タンパク質は臨床上の疲状出現時期に一致して見られ、病態把握に有用であった。
７．インフルエンザ脳症患者群の髄液からは、１４−３−３アイソフォームとしてβ、ζが検出され、頭部ＭＲＩからはヒトの脳では側頭葉と後頭葉が障害されていることが明らかになった。
本発明により、髄液中１４−３−３タンパク質を早期に検出するキットが製造できる。すなわち１４−３−３タンパク質の抗原を検出するためのキットにより、発症早期に採取された髄液を反応させて、陽性例をインフルエンザ脳症と診断できる。The main points of this research result are as follows.
1.14-3-3 protein was detected in all cases in the cerebrospinal fluid of patients with influenza encephalopathy, and none was detected in consciousness disorders such as febrile convulsions associated with high fever of influenza infection. This clearly shows that detection of 14-3-3 protein in cerebrospinal fluid is useful for early diagnosis of influenza encephalopathy.
Similarly, 2.14-3-3 protein was not detected in acute encephalopathy associated with other than influenza infection, and was useful for clearly diagnosing influenza encephalopathy among acute encephalopathy.
3. From the cerebrospinal fluid of the influenza encephalopathy patient group, β, γ, ζ, θ, ε were detected as 14-3-3 isoforms, and it was revealed that the human brain was extensively damaged.
The strong detection in the order of β, θ, γ, ζ, and ε among the 4.5 isoforms is considered to be a specific isoform appearance pattern in influenza encephalopathy.
5). Identification of the appearance pattern of these five 14-3-3 isoforms enables identification of damaged brain regions and is useful for elucidating the mechanism by which 14-3-3 protein is detected in cerebrospinal fluid. is there.
6). In mitochondrial disease (MELAS), 14-3-3 protein in cerebrospinal fluid was found in accordance with the clinical appearance of fatigue and was useful for understanding the pathological condition.
7). Β and ζ were detected as 14-3-3 isoforms from the cerebrospinal fluid of a group of patients with influenza encephalopathy, and head and MRI revealed that the temporal and occipital lobes were impaired in the human brain. It was.
According to the present invention, a kit for early detection of 14-3-3 protein in cerebrospinal fluid can be produced. That is, by using a kit for detecting an antigen of 14-3-3 protein, a cerebrospinal fluid collected early in the onset can be reacted to diagnose a positive case as influenza encephalopathy.
本発明の検査法を利用することによって、インフルエンザ脳症の検査用キットが製造できる。また、ヒト髄液中に発現する１４−３−３タンパク質からなるマーカ、ヒト髄液中に発現する１４−３−３タンパク質に特異的に結合する抗体を含むインフルエンザ脳症の診断薬、１４−３−３タンパク質に特異的に結合する一次抗体と、この一次抗体に特異的に結合し、かつ識別標識を有する二次抗体とを含み、前記識別標識の検出により前記１４−３−３タンパク質を検出するインフルエンザ脳症の診断キット、が製造できる。 By utilizing the test method of the present invention, a test kit for influenza encephalopathy can be produced. In addition, a diagnostic agent for influenza encephalopathy comprising a marker comprising a 14-3-3 protein expressed in human cerebrospinal fluid, an antibody that specifically binds to the 14-3-3 protein expressed in human cerebrospinal fluid, -3 protein, and a secondary antibody that specifically binds to the primary antibody and has an identification label, and detects the 14-3-3 protein by detecting the identification label A diagnostic kit for influenza encephalopathy can be manufactured.
Claims (16)
、θ 、γ
、ζ 、ε
の順に強く検出された場合に、インフルエンザ脳症であると判断する請求項１記載の検査方法。 In the isoform of 14-3-3 protein, β
, Θ, γ
, Ζ, ε
The test | inspection method of Claim 1 which judges that it is influenza encephalopathy when it detects strongly in order of.
、ζ およびε
のアイソフォームである請求項４記載のマーカ。 14-3-3 protein is β, θ, γ
, Ζ and ε
The marker according to claim 4, which is an isoform of
、θ 、γ
、ζ およびεのアイソフォームのそれぞれに特異的に結合する抗体を含む請求項１１記載の診断キット。 The primary antibody is β in the 14-3-3 protein.
, Θ, γ
The diagnostic kit of claim 11, comprising an antibody that specifically binds to each of the isoforms of γ, ζ and ε.
のいずれかに記載の診断キット。 15. The primary antibody and the secondary antibody are each a monoclonal antibody.
The diagnostic kit in any one of.
The diagnostic kit according to any one of claims 11 to 15, wherein the identification label is an enzyme label or a fluorescent label.
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| PCT/JP2005/006304 WO2005100992A1 (en) | 2004-04-01 | 2005-03-31 | Mtehod of examining disease with cerebral dysfunction, protein marker to be used therein, diagnostic agent and diagnostic kit |
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| JP2004134869A JP4399593B2 (en) | 2004-04-01 | 2004-04-01 | Influenza encephalopathy test method, marker consisting of protein expressed in human cerebrospinal fluid, diagnostic agent, diagnostic kit |
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| ES2438020T3 (en) | 2005-11-10 | 2014-01-15 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Compositions and methods for the treatment of addictions and other neuropsychiatric disorders |
| JP4991847B2 (en) * | 2006-05-09 | 2012-08-01 | ザ ユニバーシティ オブ ブリティッシュ コロンビア | Dissolved protein arthritis marker |
| JP5483141B2 (en) * | 2008-04-23 | 2014-05-07 | 学校法人日本医科大学 | Distinguishing between encephalopathy-derived convulsions and fever-derived thermal convulsions |
| WO2009137832A2 (en) * | 2008-05-09 | 2009-11-12 | Duke University | Autoantibodies in the detection and treatment of cancer |
| MX359263B (en) | 2010-06-16 | 2018-09-20 | Board Of Supervisors Of Louisiana State Univ & Agricultural & Mechanical College | Compositions and methods for the treatment of addiction, psychiatric disorders, and neurodegenerative disease. |
| JP2013101047A (en) * | 2011-11-08 | 2013-05-23 | Nagasaki Univ | 14-3-3 PROTEIN γ ISOFORM SPECIFIC ELISA |
| CN110582278B (en) | 2017-03-10 | 2023-04-18 | 伊姆贝拉神经疗法公司 | Pharmaceutical composition and use thereof |
| CN109593131B (en) * | 2018-12-27 | 2021-09-03 | 南京医科大学 | Monoclonal antibody for resisting 14-3-3 eta protein and application thereof |
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| JP2002071695A (en) * | 2000-08-31 | 2002-03-12 | Meneki Seibutsu Kenkyusho:Kk | Diagnostic agent, diagnostic method and diagnostic kit for aids related brain disease |
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