JP4512828B2 - Detection marker and detection kit for pulmonary sarcoidosis and ocular sarcoidosis - Google Patents

Detection marker and detection kit for pulmonary sarcoidosis and ocular sarcoidosis Download PDF

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JP4512828B2
JP4512828B2 JP2005115173A JP2005115173A JP4512828B2 JP 4512828 B2 JP4512828 B2 JP 4512828B2 JP 2005115173 A JP2005115173 A JP 2005115173A JP 2005115173 A JP2005115173 A JP 2005115173A JP 4512828 B2 JP4512828 B2 JP 4512828B2
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克志 黒須
裕一 滝口
理 岡田
喬之 栗山
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国立大学法人 千葉大学
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本発明は肺サルコイドーシスおよび眼サルコイドーシスの検出マーカー及び検出キットに関する。 The present invention relates to detection markers and detection kits for pulmonary sarcoidosis and ocular sarcoidosis.

肺サルコイドーシスは全身性の非乾酪性肉芽腫病変形成を主張とする原因不明の疾患であるが、疾患特異的な検出マーカーとしては血清中のangiotensin
converting enzyme:以下ACEやリゾチームが報告されているが、血清中のACE陽性率は50%前後とされ、血清中のリゾチームは疾患特異性が乏しいことがいわれている。
Pulmonary sarcoidosis is a disease of unknown cause that claims systemic non-buty granuloma lesion formation, but as a disease-specific detection marker, serum angiotensin
Converting enzyme: ACE and lysozyme have been reported below, but the ACE positive rate in serum is about 50%, and it is said that serum lysozyme has poor disease specificity.

サルコイドーシスは全身性の疾患であり、眼病変(眼サルコイドーシス)から発見されることも多い。眼サルコイドーシスには特徴的な眼病変が揃い、眼サルコイドーシスが強く疑われるが、全身検査陽性所見が乏しく、診断基準を満たさずに疑診にとどまる症例も稀ではない。   Sarcoidosis is a systemic disease and is often found from ocular lesions (ocular sarcoidosis). Ocular sarcoidosis has distinctive ocular lesions and ocular sarcoidosis is strongly suspected, but there are few positive findings on systemic examination, and it is not uncommon to remain suspected without meeting diagnostic criteria.

肺サルコイドーシスの診断には気管支肺胞洗浄検査(Bronchoalveolar lavage:以下BAL)が行われている。肺サルコイドーシスにおいては、BAL液中に多くの炎症細胞、とりわけCD4陽性リンパ球の増加が確認されたことの報告があり、肺胞腔や間質に浸潤したCD4 陽性リンパ球が病態に重要な役割を果たしているのではないかとの示唆がある(例えば下記非特許文献1参照)。   For diagnosis of pulmonary sarcoidosis, a bronchoalveolar lavage test (BAL) is performed. In pulmonary sarcoidosis, it has been reported that an increase in many inflammatory cells, especially CD4 positive lymphocytes, has been confirmed in the BAL fluid. There is a suggestion that this may be achieved (see, for example, Non-Patent Document 1 below).

ところで、SEREX(Sserological analysis of recombinant cDNA expression libraries)法は、約1万個のcDNAの中で数個程度しか存在しない自己抗原発現cDNAに対しても検出が可能であり、微量の自己抗原の検出に適した方法である。このSEREX法により肺癌をはじめとする様々な腫瘍関連抗原が同定されている。同定された抗原には、転写因子、細胞の分化抗原、細胞構成蛋白などに対する自己抗体に加え、新規遺伝子も含まれている。即ちSEREX法は自己抗原検索においては強力で有用な手法であり、これを用いた報告として、サルコイドーシス関連自己抗体の検索に関して一文献のみ報告がある(下記非特許文献2参照)。   By the way, the SEREX (Serologic analysis of recombinant cDNA expression libraries) method can detect even a few self-antigen-expressing cDNAs out of about 10,000 cDNAs, and detect a small amount of self-antigens. This is a suitable method. Various tumor-related antigens including lung cancer have been identified by this SEREX method. The identified antigens include novel genes in addition to autoantibodies against transcription factors, cell differentiation antigens, cell constituent proteins, and the like. In other words, the SEREX method is a powerful and useful technique in the search for self-antigen, and as a report using this method, there is only one document regarding the search for sarcoidosis-related autoantibodies (see Non-Patent Document 2 below).

PCR(Polimerase Chain Reaction)法によるBAL液中のT細胞Vα/β鎖遺伝子再構成の検索では、抗原特異的なT細胞増生が肺サルコイドーシスに認められ、HLA クラスII 抗原の抗原収容溝に収まった抗原ペプチドとHLA 抗原の複合体と適切に結合する構造を持ったT細胞抗原受容体を有するT細胞レパトリーが抗原特異的免疫反応によって増殖したことを意味し、疾患関連抗原の存在が示唆されている(例えば下記非特許文献3参照)。   In the search for T cell Vα / β chain gene rearrangement in BAL fluid by PCR (Polymerase Chain Reaction) method, antigen-specific T cell proliferation was observed in pulmonary sarcoidosis, and was contained in the antigen-containing groove of HLA class II antigen This means that a T cell repertoire having a T cell antigen receptor having a structure that appropriately binds to a complex of an antigen peptide and an HLA antigen has proliferated by an antigen-specific immune reaction, suggesting the presence of a disease-related antigen. (See, for example, Non-Patent Document 3 below).

Gernot Z ら、”TCR Vβ Families in T cell clones from sarcoid lung parenchyma, BAL, and blood”、Am J Respir Crit Care Med 1997年、156巻、1593−1600頁Gernot Z et al., “TCR Vβ Family in T cell clones from sarcoid lun parenchyma, BAL, and blood”, Am J Respir Crit Care Med, 1997, pp. 159-153. Ebeら、“Proliferative response of peripheral blood mononuclear cells and levels of antibody to recombinant protein from Propionibacterium acnes DNA expression library in Japanese patients with sarcoidosis”、Sarcoidosis Vasc Diffuse Lung Dis 2000年、17巻、256−265頁Ebe et al., "Proliferative response of peripheral blood mononuclear cells and levels of antibody to recombinant protein from Propionibacterium acnes DNA expression library in Japanese patients with sarcoidosis", Sarcoidosis Vasc Diffuse Lung Dis 2000 years, Vol. 17, pp. 256-265 Dohi Mら、” Accumulation of multiple T cell clonotypes in lungs of healthy individuals and patients with pulmonary sarcoidosis”、J Immunol 1994年、152巻、1983−1988頁Dohi M et al., “Accumulation of multiple T cell clonotypes in lungs of health individuals and patients with pulmonary sarcoidosis, 198, 152, J Immun.

確かに、上記非特許文献1においてCD4陽性T細胞が何らか病態に関与していることを示唆しているが、CD4陽性T細胞が増加する機序や網膜炎が合併する機序は大部分が不明であり、しかも、末梢血ACEの増加のみによっては肺サルコイドーシスの診断を行うことが困難な症例が多数存在する。眼科医が発見する眼病変自体の大きさは数十ミクロンから数ミリ単位以下であるため、病巣が眼内に限局する場合には、眼内病巣からACE などが流血中に流出しても、全身単位での活性はきわめて低くなり、通常の検査では検出限度以下となる。眼サルコイドーシスに特異的な疾患マーカーは現在存在しないため、ぶどう膜炎を初発症状としても、眼サルコイドーシスの診断がきわめて困難である。   Certainly, the above Non-Patent Document 1 suggests that CD4-positive T cells are involved in some pathological condition, but most of the mechanisms by which CD4-positive T cells increase and retinitis are combined. There are many cases in which it is difficult to diagnose pulmonary sarcoidosis only by increasing peripheral blood ACE. Since the size of the ocular lesion detected by an ophthalmologist is several tens of microns to several millimeters or less, if the lesion is confined in the eye, even if ACE or the like flows from the intraocular lesion into the bloodstream, The activity in the whole body unit is extremely low, and it is below the detection limit in a normal test. Since there is currently no disease marker specific to ocular sarcoidosis, it is very difficult to diagnose ocular sarcoidosis even if uveitis is the initial symptom.

また、SEREX法については、殆どが腫瘍関連抗原の同定のために用いられているものであって、腫瘍関連抗原以外のものに適用した例は極めて稀である。これを用いたサルコイドーシスの報告としては上記非特許文献2の一文献のみ、報告があるが、この報告は、Propionibacterium acnes 菌DNAを組み込んだファージを用いたSEREX解析であり、肺胞上皮を含め人の細胞が発現するcDNAを組み込んだファージを用いたSEREX解析は行われていない。   Moreover, most of the SEREX methods are used for identification of tumor-associated antigens, and there are very few examples where they are applied to other than tumor-associated antigens. There is a report of sarcoidosis using this in only one document of Non-Patent Document 2 described above. This report is a SEREX analysis using a phage incorporating Propionibacterium acnes DNA, and includes humans including alveolar epithelium. SEREX analysis using a phage into which cDNA expressed by the cells of FIG.

更に、上記非特許文献3に記載の報告では、抗原特異的なT細胞増生が認められているが、PCR法のみでは症例間でHLA抗原が異なること、認識している抗原は抗原提示細胞によって断片化されていること、からサルコイドーシス関連自己抗体が認識する抗原の決定は極めて困難である。   Furthermore, in the report described in Non-Patent Document 3 above, antigen-specific T cell proliferation is recognized, but the HLA antigen differs between cases only by the PCR method, and the recognized antigen depends on the antigen-presenting cell. Since it is fragmented, it is extremely difficult to determine the antigen recognized by the sarcoidosis-related autoantibody.

そこで、本発明は、上記課題を鑑み、肺サルコイドーシスおよび眼サルコイドーシスを検出するための検出マーカー及び検出キットを提供することを目的とする。   In view of the above problems, an object of the present invention is to provide a detection marker and a detection kit for detecting pulmonary sarcoidosis and ocular sarcoidosis.

BAL液中や肺間質中に浸潤するT細胞は、その免疫グロブリン可変部領域にVα/Vβ鎖を有しており、肺胞局所に疾患特異的な抗原が存在し、肺において疾患特異的な抗原/抗体反応が生じているのであれば、B細胞による抗体産生にはCD4陽性T細胞の援助が必要不可欠である。まず、我々も肺サルコイドーシス症例のBAL液中のCD4陽性T細胞のVβ鎖を解析したところ、全例に特定のCD4陽性T細胞のoligoclonalな増生が認められた。この所見はいままでの肺サルコイドーシスのBAL液における報告を裏付け、肺胞局所において特異的な抗原に対してCD4陽性T細胞が増生し、活発な抗原抗体反応の存在を示唆する結果であった。眼サルコイドーシスに伴って縦隔リンパ節腫脹や肺内陰影が合併することも稀ではなく、肺胞と眼において共通の自己抗原に対する免疫反応が生じている可能性も考えられる。もしそのような自己抗原が存在するのであれば、眼周囲のリンパ節腫脹はほとんど認められないことと肺サルコイドーシスが合併する場合には縦隔リンパ節腫脹を併発していることが多いことから、サルコイドーシスに伴う抗原刺激は肺内に強く存在していることが示唆される。   T cells that infiltrate into the BAL fluid or lung interstitium have Vα / Vβ chains in their immunoglobulin variable region, and have disease-specific antigens in the alveoli, which are disease-specific in the lungs. If an antigen / antibody reaction occurs, the assistance of CD4 positive T cells is indispensable for antibody production by B cells. First, we also analyzed the Vβ chain of CD4 positive T cells in BAL fluid of pulmonary sarcoidosis cases, and in all cases, specific proliferation of specific CD4 positive T cells was observed. This finding supported the previous reports of pulmonary sarcoidosis in the BAL fluid, and resulted in the proliferation of CD4-positive T cells against a specific antigen in the alveolar region, suggesting the existence of an active antigen-antibody reaction. It is not uncommon for mediastinal lymphadenopathy and intrapulmonary shadow to accompany ocular sarcoidosis, and it is possible that an immune response to a common self-antigen occurs in the alveoli and the eye. If such autoantigens are present, there is almost no periocular lymphadenopathy, and if pulmonary sarcoidosis is complicated, mediastinal lymphadenopathy often accompanies. It is suggested that antigen stimulation accompanying sarcoidosis is strongly present in the lung.

そこで、更に本発明者らが上記について検討を行ったところ、肺胞内腔にはその構成細胞として、 II型肺胞上皮と肺胞マクロファージがあげられる。肺胞腔内でサルコイドーシス特異的な抗原抗体反応が生じているのであれば、構成細胞として大部分を占める肺胞上皮やマクロファージが有する蛋白が抗原として働いているのではないかと推察した。肺胞上皮が発現する蛋白を非常に鋭敏な方法(SEREX法)で解析を行うことで、肺サルコイドーシスの患者血清中に存在する自己抗体の検出が可能であることを見いだした。以下具体的に示すが、我々の発見した9種類の自己抗体は比較的高頻度で肺サルコイドーシス症例の血清中に存在し、これらのうちのひとつであるretinal short−chain dehydrogenase/reductase (ret SDR2) とtrabecular meshwork構成蛋白(accession number:CD676906, CD677025, CD677068)、ocular pericyte 構成蛋白(CD066293)、網膜構成蛋白(BQ637781)、レンズ構成蛋白(CD674,39)は肺胞上皮と網膜に共通に存在する蛋白であることから、肺胞腔内で肺胞上皮やマクロファージから流出した自己抗原蛋白が抗原となって抗体産生が生じ、この蛋白は網膜にも発現している蛋白のため、これらの異常な自己抗体が眼サルコイドーシスの眼病変の病因の一因であり、これらの自己抗体の測定は眼サルコイドーシスの診断に有用であることを発見した。これらを組み合わせることによって、肺サルコイドーシスおよび眼サルコイドーシスの正確な血清学的診断が可能となる。   Then, when the present inventors further examined the above, the alveolar lumen includes type II alveolar epithelium and alveolar macrophages as its constituent cells. If a sarcoidosis-specific antigen-antibody reaction occurred in the alveolar space, it was inferred that the proteins of alveolar epithelium and macrophages, which occupy most of the constituent cells, acted as antigens. It was found that autoantibodies present in sera of patients with pulmonary sarcoidosis can be detected by analyzing proteins expressed by alveolar epithelium by a very sensitive method (SEREX method). As specifically shown below, the nine types of autoantibodies we discovered are present in the serum of pulmonary sarcoidosis cases with relatively high frequency, one of which is retinal short-chain dehydrogenase / reductase (ret SDR2). And trabecular meshwork constituent protein (accession number: CD676906, CD677025, CD677068), ocular pericite constituent protein (CD066293), retinal constituent protein (BQ637781), and lens constituent protein (CD674,39) are common to alveolar epithelium and retina Because it is a protein, self-antigen protein that flows out of the alveolar epithelium and macrophages in the alveolar cavity is used as an antigen to produce antibodies, and this protein is also present in the retina. For that represents protein, these abnormal autoantibodies are contributing to the pathogenesis of ocular lesions of the eye sarcoidosis found that measurement of these autoantibodies is useful in the diagnosis of eye sarcoidosis. By combining these, an accurate serological diagnosis of pulmonary sarcoidosis and ocular sarcoidosis becomes possible.

即ち、本発明に係る肺サルコイドーシスの検出マーカーは、抗poly a binding protein 1抗体、抗1−phosphatidylinositol−4−phosphate 5 kinase isoform c抗体、抗retinal short−chain dehydrogenase/reductase (ret SDR2)抗体、抗protein synthesis initiation factor 4a−II抗体、抗S−adenosylhomocysteine hydrolase抗体、抗macrophage migration inhibitory factor抗体、抗cytochrome c reductase core protein抗体、抗oxidoreductase protein抗体、抗transforming growth factor beta 2抗体の少なくとも一つを含有してなることを特徴とする。また、眼サルコイドーシスの検出マーカーは、抗retinal short−chain dehydrogenase/reductase (ret SDR2)抗体、抗retinal short−chain dehydrogenase/reductase(ret SDR2)抗体、抗trabecular meshwork構成蛋白(accession number: CD676906,CD677025,CD677068)抗体、抗ocular pericyte 構成蛋白(CD066293)抗体、抗網膜構成蛋白(BQ637781)抗体、抗レンズ構成蛋白(CD674,39)抗体の少なくとも一つを含有してなることを特徴とする。   That is, the detection marker of pulmonary sarcoidosis according to the present invention includes anti-poly binding protein 1 antibody, anti-1-phosphatidylinositol-4-phosphate 5 kinase isoformc antibody, anti-retinshot-chain dehydrogenase antibody, protein synthesis initiation factor 4a-II antibody, anti-S-adenosylhomocysteine synthetic antibody, anti-macrophage migration inhibitory factor antibody, anti-cytoreductive antibody It comprises at least one of a ccase protein antibody and an anti-transforming growth factor beta 2 antibody. In addition, detection markers for ocular sarcoidosis are anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibody, anti-retinal short-chain dehydrase / bacterial protein 70 (reverse SDR2) antibody, anti-retinal short-chain dehydrogenase It comprises at least one of a CD677068) antibody, an anti-occlusive peritite constituent protein (CD066293) antibody, an anti-retinal constituent protein (BQ637781) antibody, and an anti-lens constituent protein (CD674,39) antibody.

また、本発明に係る肺サルコイドーシスの検出キットは、poly a binding protein 1、1−phosphatidylinositol−4−phosphate 5 kinase isoform c、 retinal short−chain dehydrogenase/reductase (ret SDR2) 、 protein synthesis initiation factor 4a−II、s−adenosylhomocysteine hydrolase、 macrophage migration inhibitory factor、cytochrome c reductase core protein 、 oxidoreductase protein 、 transforming growth factor beta 2、の少なくともいずれかの抗原タンパクを基材に吸着させてなることを特徴とする。また、眼サルコイドーシスの検出キットは、retinal short−chain dehydrogenase/reductase (ret SDR2)、trabecular meshwork構成蛋白(accession number: CD676906, CD677025, CD677068)、ocular pericyte 構成蛋白(CD066293)、網膜構成蛋白(BQ637781)、レンズ構成蛋白(CD674,39)の少なくともいずれかの抗原タンパクを基材に吸着させてなることを特徴とする。なおここで基材としては上記抗原タンパクを吸着させて抗体反応を確認できる限りにおいて限定はされないが、メンブレンやELISA法にも散られるプレートなどが該当する。またこの場合において、抗原タンパクは標識が付されていること、標識は、His−tag標識が付されていること、も望ましい。   In addition, the detection kit for pulmonary sarcoidosis according to the present invention includes poly a binding protein 1, 1-phosphotylated-lintositol 4-phosphonate 5 kinase isoform, c, retinshort re-in 2 , S-adenosyl homosteinine hydrolase, macromigration migration factor, cytochrome product core protein, oxidoreductase protein protein, It is characterized by adsorbing at least one antigen protein of forming growth factor beta 2 to a substrate. In addition, detection kits for ocular sarcoidosis include retinal short-chain dehydrogenase / reductase (ret SDR2), trabecular meshwork protein (accession number: CD6760p, CD6767025, CD677025, CD667025, CD667025, CD677025, Further, it is characterized in that at least one antigen protein of the lens constituent protein (CD674, 39) is adsorbed on a base material. Here, the substrate is not limited as long as the antigen protein can be adsorbed and the antibody reaction can be confirmed, but includes a membrane and a plate scattered in the ELISA method. In this case, it is also desirable that the antigen protein is labeled, and the label is labeled with a His-tag label.

また、本発明に係る方法としては、poly a binding protein 1、 1−phosphatidylinositol−4−phosphate 5 kinase isoform c、 retinal short−chain dehydrogenase/reductase (ret SDR2) 、 protein synthesis initiation factor 4a−II、s−adenosylhomocysteine hydrolase、 macrophage migration inhibitory factor、cytochrome c reductase core protein 、 oxidoreductase protein 、 transforming growth factor beta 2、trabecular meshwork構成蛋白(accession number: CD676906, CD677025, CD677068)、ocular pericyte 構成蛋白(CD066293)、網膜構成蛋白(BQ637781)、レンズ構成蛋白(CD674,39)の少なくともいずれかの標識された抗原タンパクを吸着したプレートに血液又はBAL液を塗布することを特徴とする。   In addition, as a method according to the present invention, poly a binding protein 1, 1-phosphatidylinositol-4-phosphate 5 kinase isoform c, retinal short-chain dehydrogenase / reducetitereduction adenosyl homosteinine hydrolase, macromigration migration factor, cytochrome reductase core protein, oxidoreductase protein complex low factor beta 2, trabecular meshwork constituent protein (accession number: CD676906, CD677025, CD677068), ocular pericite constituent protein (CD066633), retinal constituent protein (BQ637781), lens constituent protein CD3967 It is characterized in that blood or BAL solution is applied to a plate adsorbed with the antigen protein.

以上、肺サルコイドーシスおよび眼サルコイドーシスを検出するための検出マーカー及び検出キットを提供することができるようになる。特に、今回我々の発見した15種類の自己抗体は既存には報告されておらず、サルコイドーシス以外の症例ではほとんどの自己抗体は検出されず、診断検査としての利用価値が高い。   As described above, a detection marker and a detection kit for detecting pulmonary sarcoidosis and ocular sarcoidosis can be provided. In particular, the 15 types of autoantibodies we have discovered have not been reported in the past, and most autoantibodies are not detected in cases other than sarcoidosis, which is highly useful as a diagnostic test.

本発明を用いることによって、治療前後のBAL液または経時的に採取した血清による連続的な疾患特異的自己抗体の解析が可能となる。とりわけ、His−tag標識自己抗原蛋白を用いた自己抗体の測定は、病勢フォローやステロイドなどの治療効果判定に最適な検査法となる。   By using the present invention, it is possible to continuously analyze disease-specific autoantibodies using BAL fluid before and after treatment or serum collected over time. In particular, measurement of autoantibodies using His-tag labeled autoantigen protein is an optimal test method for disease follow-up and determination of therapeutic effects such as steroids.

以下、本発明の実施形態について図面を用いて説明する。   Hereinafter, embodiments of the present invention will be described with reference to the drawings.

本発明の実施の形態としては、肺サルコイドーシスおよび眼サルコイドーシスの検出マーカー、検出キットの態様を採用できる。肺サルコイドーシス検出マーカーの態様として、抗poly a binding protein 1抗体、抗1−phosphatidylinositol−4−phosphate 5 kinase isoform c抗体、抗 retinal short−chain dehydrogenase/reductase (ret SDR2) 抗体、抗 protein synthesis initiation factor 4a−II抗体、抗 s−adenosylhomocysteine hydrolase抗体、抗 macrophage migration inhibitory factor抗体、抗cytochrome c reductase core protein 抗体、抗 oxidoreductase protein 抗体、抗 transforming growth factor beta 2抗体の少なくとも一つを含んでなることとする。眼サルコイドーシスの検出マーカーの態様として抗 retinal short−chain dehydrogenase/reductase (ret SDR2) 抗体、抗 retinal short−chain dehydrogenase/reductase (ret SDR2) 抗体、抗trabecular meshwork構成蛋白(accession number: CD676906, CD677025, CD677068)抗体、抗ocular pericyte 構成蛋白(CD066293)抗体、抗網膜構成蛋白(BQ637781)抗体、抗レンズ構成蛋白(CD674,39)抗体の少なくとも一つを含んでなることとする。即ち、肺サルコイドーシスまたは眼サルコイドーシスの疑いのある患者から血液又はBAL液を抽出し、この抗体が存在しているか否かを調べ、存在している場合は肺サルコイドーシスまたは眼サルコイドーシスの疑いがあると判定することができる。これにより精度よく調べることができるようになる。   As an embodiment of the present invention, a detection marker and detection kit of pulmonary sarcoidosis and ocular sarcoidosis can be employed. Examples of pulmonary sarcoidosis detection markers include anti-poly a binding protein 1 antibody, anti-1-phosphophatidylinositol-4-phosphate 5-kinase isoform antibody, anti-retinshot / retin anti-DR2 -II antibody, anti-s-adenosyl homocysteine hydrochloride antibody, anti-macrophage migration inhibitory factor antibody, anti-cytochrome reductase core protein antibody, anti-oxidorducuc It is supposed to comprise at least one of a case protein antibody and an anti-transforming growth factor beta 2 antibody. Anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibody, anti-retin short-chain 70 gene, anti-retinsh-chain 70-antibody ) Antibody, anti-occlusive peritite constituent protein (CD066293) antibody, anti-retinal constituent protein (BQ637781) antibody, and anti-lens constituent protein (CD674,39) antibody. That is, blood or BAL fluid is extracted from a patient suspected of having pulmonary sarcoidosis or ocular sarcoidosis, and the presence or absence of this antibody is examined. If present, it is determined that pulmonary sarcoidosis or ocular sarcoidosis is suspected. can do. This makes it possible to investigate with high accuracy.

また本発明の一実施形態として、poly a binding protein 1、1−phosphatidylinositol−4−phosphate 5 kinase isoform c、 retinal short−chain dehydrogenase/reductase (ret SDR2) 、 protein synthesis initiation factor 4a−II、 s−adenosylhomocysteine hydrolase、 macrophage migration inhibitory factor、cytochrome c reductase core protein 、 oxidoreductase protein 、 transforming growth factor beta 2、trabecular meshwork構成蛋白(accession number: CD676906, CD677025, CD677068)、ocular pericyte 構成蛋白(CD066293)、網膜構成蛋白(BQ637781)、レンズ構成蛋白(CD674,39)、の少なくともいずれかの抗原タンパクを基材に吸着させてなる肺サルコイドーシスおよび眼サルコイドーシスの検出キットとすることもできる。これにより、肺サルコイドーシスまたは眼サルコイドーシスの疑いのある患者から血液又はBAL液を抽出し、この抗原タンパクが抗体と反応しているか否かを調べることにより、肺サルコイドーシスまたは眼サルコイドーシスの疑いがあるか否かを判定することができる。具体的にはこれら抗原タンパクを基材に吸着させ、BAL液又は血清などを塗布することにより抗原タンパクと反応させ、更に発光又は発色部位を有する二次抗体と反応させることで、発光等が行われるか、又はその量にもとづいて抗体が存在しているか否かを判定し、肺サルコイドーシスおよび眼サルコイドーシスであるか否かを判定することが可能となる。この場合において、抗原タンパクには抗原タンパクを作成する観点から標識が付されていることが望ましい。特に本実施形態によると、あらかじめ作成した自己抗原蛋白が吸着したメンブレンやプレートを利用することによって、迅速な検査が可能であり、1時間程度で結果を得ることができる。そのため、病状が刻々と変化する重症例のサルコイドーシス症例においても迅速な対応が可能である。肺サルコイドーシスが悪化した場合には、できるだけ早い治療が必要であるが、他の合併症(感染症、悪性腫瘍)の除外に時間がかかり、診断に苦慮する場合が多い。本発明は肺サルコイドーシス自体が悪化したのか他の合併症が重なったのかを鑑別するうえで非常に重要な情報を提供可能であり、迅速な対応に結びつくことができる。   In addition, as an embodiment of the present invention, poly a binding protein 1, 1-phosphotylino sito 4- phosphatate 5 kinase isoform form c hydrolase, macromigration migration factor, cytochrome reductase core protein, oxidoreductase protein, transforming gr wth factor beta 2, trabecular meshwork constituent protein (accession number: CD676906, CD677025, CD677068), ocular pericite constituent protein (CD066633), retinal constituent protein (BQ637781), lens constituent protein CD (67) A kit for detecting pulmonary sarcoidosis and ocular sarcoidosis, wherein a protein is adsorbed on a substrate, can also be used. Whether blood or BAL fluid is extracted from a patient suspected of having pulmonary sarcoidosis or ocular sarcoidosis, and whether or not this antigenic protein reacts with an antibody to determine whether suspicion of pulmonary sarcoidosis or ocular sarcoidosis is present. Can be determined. Specifically, these antigen proteins are adsorbed on a substrate, reacted with an antigen protein by applying a BAL solution or serum, etc., and further reacted with a secondary antibody having a luminescence or color development site, thereby causing luminescence or the like. It is possible to determine whether or not the antibody is present based on the amount of the pulmonary sarcoidosis and ocular sarcoidosis. In this case, it is desirable that the antigen protein is labeled from the viewpoint of producing the antigen protein. In particular, according to the present embodiment, by using a membrane or plate adsorbed with a self-antigen protein prepared in advance, rapid examination is possible, and a result can be obtained in about one hour. Therefore, it is possible to promptly respond to severe sarcoidosis cases whose medical condition changes every moment. When pulmonary sarcoidosis worsens, it is necessary to treat it as soon as possible, but it often takes time to exclude other complications (infections, malignant tumors) and is difficult to diagnose. The present invention can provide very important information in distinguishing whether the pulmonary sarcoidosis itself has deteriorated or other complications, and can lead to a prompt response.

以下、本実施形態に係る発明の効果を実証すべく検討を行った。以下説明する。   In the following, studies were conducted to verify the effects of the invention according to this embodiment. This will be described below.

T細胞Vβ鎖は25種類のサブファミリーから構成されているため、まずそれぞれのサブファミリーに特異的なプライマーを作成した。作成したプライマーを図1に示す。なお図中、Vβ1、Vβ2…、Vβ25はfowardとして用い、Cβはreverseとして共通に用いた。   Since the T cell Vβ chain is composed of 25 types of subfamilies, first, primers specific to each subfamily were prepared. The prepared primer is shown in FIG. In the figure, Vβ1, Vβ2,..., Vβ25 are used as forwards, and Cβ is commonly used as reverse.

そして肺サルコイドーシス20症例に対しBALを行い、そのBALを抽出した。そしてこのBAL液からDyna beadsを用いてCD4陽性T細胞の分離、total RNAの抽出、RT−PCR法によるT細胞のVβ鎖可変部領域の増幅を行い、更にこのPCR産物を2%アガロースで泳動しエチジウムブロマイドによる染色では、ごく一部のVβ鎖サブグループのみが発現していた(図2参照)。   BAL was performed on 20 cases of pulmonary sarcoidosis, and the BAL was extracted. Then, CD4 positive T cells are separated from this BAL solution using Dyna beads, total RNA is extracted, the Vβ chain variable region of T cells is amplified by RT-PCR, and this PCR product is electrophoresed with 2% agarose. By staining with ethidium bromide, only a small part of the Vβ chain subgroup was expressed (see FIG. 2).

次に、SEREX法の概要を図3に示す。II型肺胞上皮癌培養株からmRNAを抽出し、cDNAライブラリーを作成後、得られたcDNA ライブラリーを蛋白発現ベクター(ZAP発現ベクター)に組み込み作成したファージを大腸菌に感染させ、c−DNAのコードする蛋白を大腸菌に発現させた。そしてcolony hybridization法によって、肺サルコイドーシスの血清およびBAL液中の免疫グロブリンと結合するプラークを選別し、陽性ファージをファジェミドベクターに変換後、シークエンスによって特異的蛋白の塩基配列を決定した。ホモロジー検索によって蛋白の構造を決定し、肺サルコイドーシス/眼サルコイドーシスの血清中およびBAL液中に存在する自己抗体の認識自己抗原の同定を行った。以上の結果、症例とその症例に対して検出された自己抗体が認識している抗原を図4、5に示す。   Next, an outline of the SEREX method is shown in FIG. MRNA is extracted from a type II alveolar epithelial cancer culture, and a cDNA library is prepared. The resulting cDNA library is incorporated into a protein expression vector (ZAP expression vector) and infected with Escherichia coli. The protein encoded by was expressed in E. coli. Then, plaques that bind to immunoglobulin in pulmonary sarcoidosis serum and BAL fluid were selected by colony hybridization, and positive phages were converted into phagemid vectors, and then the base sequences of specific proteins were determined by sequencing. The structure of the protein was determined by homology search, and autoantibodies recognized in the serum and BAL fluid of pulmonary sarcoidosis / ocular sarcoidosis were identified. As a result of the above, FIGS. 4 and 5 show the antigens recognized by the cases and the autoantibodies detected for the cases.

図4、図5によると、15種類の抗体のうち、抗poly a binding protein 1抗体は、SEREX法によって解析した肺サルコイドーシス10例中5例(50%)の血清および BAL液中に存在した。抗1−phosphatidylinositol−4−phosphate 5 kinase isoform c抗体と抗 retinal short−chain dehydrogenase/reductase (ret SDR2) 抗体は肺サルコイドーシス10例中4例(40%)の血清およびBAL液中に存在していた。抗 protein synthesis initiation factor 4a−II抗体は肺サルコイドーシス10例中3例(30%)の血清およびBAL液中に存在していた。抗 s−adenosylhomocysteine hydrolase抗体、抗 macrophage migration inhibitory factor抗体、抗cytochrome c reductase core protein 抗体、抗 oxidoreductase protein 抗体、抗 transforming growth factor beta 2抗体、抗trabecular meshwork構成蛋白(accession number: CD676906, CD677025, CD677068)抗体、抗ocular pericyte 構成蛋白(CD066293)抗体、抗網膜構成蛋白(BQ637781)抗体、抗レンズ構成蛋白(CD674,39)抗体は10例中1例(10%)に存在していた。これにより、自己抗体を検出することによって、肺サルコイドーシス、眼サルコイドースの有無を判定することができることを確かめた。   According to FIG. 4 and FIG. 5, anti-poly binding protein 1 antibody was present in the serum and BAL fluid of 5 cases (50%) out of 10 cases analyzed by SEREX method among 15 types of antibodies. Anti-1-phosphatidylinoside-4-phosphate 5 kinase isoform c antibody and anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibodies were present in 4 cases (40%) in 10 cases of pulmonary sarcoidosis and BAL (40%). . Anti-protein synthesis initiation factor 4a-II antibody was present in the serum and BAL fluid of 3 (30%) of 10 pulmonary sarcoidosis cases. Anti s-adenosylhomocysteine hydrolase antibody, anti-macrophage migration inhibitory factor antibodies, anti-cytochrome c reductase core protein antibody, anti-oxidoreductase protein antibody, anti-transforming growth factor beta 2 antibodies, anti-trabecular meshwork structure protein (accession number: CD676906, CD677025, CD677068) Antibody, anti-occlusive peritite constituent protein (CD066293) antibody, anti-retinal constituent protein (BQ637781) antibody, anti-lens constituent protein (CD674,39) antibody is 1 in 10 cases It was present in 10%). Thus, it was confirmed that the presence or absence of pulmonary sarcoidosis and ocular sarcoidose can be determined by detecting autoantibodies.

また今回SEREX法で解析した10例中4例 (図6)はACEの上昇認めるが、今回発見した抗体のうち陽性率の高い4つの自己抗体(抗poly
a binding protein 1抗体、抗1-phosphatidylinositol-4-phosphate
5 kinase isoform c抗体、抗 retinal short-chain dehydrogenase/reductase
(ret SDR2) 抗体、抗protein synthesis initiation factor 4a−II抗体)を組あわせることによって、10例中9例(90%)は、新規自己抗体が陽性であり、ACEが陰性であった6例はすべて自己抗体が陽性であった。肺胞と網膜の両方に発現がみられる共通抗原のうち、今回検出頻度が高かった抗 retinal short-chain dehydrogenase/reductase (ret SDR2) 抗体は、眼サルコイドーシス症例7例中3例(43%)に陽性であった。また抗 retinal short-chain dehydrogenase/reductase (ret SDR2) 抗体が陽性で現在眼サルコイドーシスの症状を有していない残りの1例においても、ガリウムシンチ検査では眼に集積が認められ、眼にもサルコイドーシス病変が潜在している可能性が示唆された。肺胞と眼の共通抗原に対する自己抗体とくに抗 retinal short-chain dehydrogenase/reductase (ret SDR2) 抗体は眼サルコイドーシスの診断マーカーとして有用である。
In 4 cases (Fig. 6) of the 10 cases analyzed by the SEREX method, an increase in ACE was observed, but among the antibodies found this time, four autoantibodies (anti-poly) with a high positive rate were observed.
a binding protein 1 antibody, anti-1-phosphatidylinositol-4-phosphate
5 kinase isoform c antibody, anti-retinal short-chain dehydrogenase / reductase
(ret SDR2) antibody, anti-protein synthesis initiation factor 4a-II antibody), 9 out of 10 cases (90%) were positive for new autoantibodies and 6 cases were negative for ACE All were autoantibodies positive. Of the common antigens that are expressed in both the alveoli and the retina, anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibodies, which were detected frequently this time, were present in 3 (43%) of 7 ocular sarcoidosis cases. It was positive. In the remaining 1 patient who is positive for anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibody and currently has no symptoms of ocular sarcoidosis, accumulation in the eye was observed by gallium scintigraphy, and sarcoidosis lesions were also observed in the eye. It is suggested that there is a potential. Autoantibodies against alveolar and ocular common antigens, especially anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibodies, are useful as diagnostic markers for ocular sarcoidosis.

また、今回検出した15種類の自己抗原蛋白のうち発現頻度が高い3種類の蛋白(poly a binding protein a 1、1−phosphatidylinositol−4−phosphate 5 kinase isoform c、 retinal short−chain dehydrogenase/reductase (ret SDR2))について、自己抗体認識抗原遺伝子全体に対するプライマーを作成し、RT−PCR法で解析し、それぞれの遺伝子の発現を確認した。図7にここで設計したプライマーを示す。3種類のうち2種類(poly a binding protein a 1、retinal short−chain dehydrogenase/reductase (ret SDR2))の自己抗原蛋白は、II型肺胞上皮癌培養株(A549)および単球系培養株(THP−1)の双方に対して発現を確認することができた。残りの1種類の自己抗原蛋白(1−phosphatidylinositol−4−phosphate 5 kinase isoform c)は、II型肺胞上皮癌培養株(A549)のみに発現が認められた。肺サルコイドーシスは肺胞腔内に病変の主座が存在すると考えられており、肺胞腔側の構成細胞の大部分を占めるII型肺胞上皮および肺胞マクロファージの両者から自己抗体認識抗原蛋白が産生されている可能性を確認した。この結果を図8に示す。そして更に、発見した自己抗体認識抗原蛋白をHis−tag標識蛋白として人工的に大腸菌に作成させた。以下に行った蛋白作成方法の概要を示す。   In addition, among the 15 types of autoantigen proteins detected this time, three types of proteins (poly a binding protein a 1, 1-phosphatidylinositol-4-phosphate 5-kinase isoform c, retinal short-chain dehydrogenase / For SDR2)), primers for the whole autoantibody-recognizing antigen gene were prepared and analyzed by RT-PCR to confirm the expression of each gene. FIG. 7 shows the primers designed here. Two types of autoantigen proteins (poly a binding protein a 1, retin short-chain dehydrogenase / reductase (ret SDR2)) are classified into type II alveolar epithelial cancer culture (A549) and monocyte culture ( Expression could be confirmed for both THP-1). Expression of the remaining one type of autoantigen protein (1-phosphatidylinositol-4-phosphate 5 kinase isoform c) was observed only in the type II alveolar epithelial cancer culture (A549). Pulmonary sarcoidosis is thought to have a lesion site in the alveolar space, and autoantibody-recognizing antigen protein is expressed from both type II alveolar epithelium and alveolar macrophages that occupy most of the constituent cells on the alveolar space side. The possibility of being produced was confirmed. The result is shown in FIG. Further, the discovered autoantibody-recognizing antigen protein was artificially prepared in Escherichia coli as a His-tag labeled protein. The outline of the protein production method performed below is shown.

上記のRT−PCR法によってで得られたPCR産物をHis−tag標識蛋白発現ベクター(Pqe−30UA)にライゲーションし、コンペテントM15細胞にトランスフォーメーションを行った。得られたHis−tag標識蛋白発現コロニー蛋白をニトロセルロースフィルターに転写後、IPTGを含むプレートにフィルターを移し6×hisタグ標識蛋白質を発現させた。得られたフィルターをPenta−HisHRP Conjugateを用いて免疫染色を行い自己抗体認識抗原蛋白発現コロニーを選別した。得られたコロニーをピックアップし培養液中で増殖させ、6×hisタグ標識蛋白質の抽出を行った。Western blot法によって蛋白発現を確認した後、大腸菌大量培養液から、自己抗体認識抗原蛋白(His−tag標識蛋白)の精製を行った。   The PCR product obtained by the above RT-PCR method was ligated to a His-tag labeled protein expression vector (Pqe-30UA) and transformed into competent M15 cells. The obtained His-tag labeled protein-expressing colony protein was transferred to a nitrocellulose filter, and then the filter was transferred to a plate containing IPTG to express a 6 × his tag labeled protein. The obtained filter was immunostained using Penta-HisHRP Conjugate to select autoantibody recognition antigen protein expression colonies. The obtained colonies were picked up and grown in a culture solution, and 6 × his tag-labeled protein was extracted. After confirming protein expression by Western blot method, autoantibody recognition antigen protein (His-tag labeled protein) was purified from E. coli mass culture.

これにより得られた15種類の肺サルコイドシス特異的His−tag標識自己抗原蛋白をThe Convertible(Biometra社)を用いて、6mm Dotとしてナイロンメンブレン上に吸着させた。これにより、得られた蛋白吸着メンブレンを症例の血清およびBAL液でhybridizationを行い、2次抗体として抗ヒトIgG抗体を結合させ化学発光にて自己抗体発現の有無を11種類同時に検出した。Dot blot法およびWestern blot法による検索では、肺サルコイドーシスの血清および気管支肺胞洗浄液を用いた場合にのみHis−tag標識蛋白に対して陽性バンドを検出することができ、またバンドの発現強度は、病勢が悪化した場合に強く検出される傾向が認められた。   The 15 types of pulmonary sarcoidosis-specific His-tag labeled autoantigen proteins thus obtained were adsorbed on a nylon membrane as 6 mm dots using The Convertible (Biometer). Thus, the obtained protein-adsorbing membrane was hybridized with the serum and BAL fluid of the case, and anti-human IgG antibody was bound as a secondary antibody, and eleven types of autoantibody expression were detected simultaneously by chemiluminescence. In the search by the dot blot method and the western blot method, a positive band for His-tag labeled protein can be detected only when lung sarcoidosis serum and bronchoalveolar lavage fluid are used. There was a tendency to be strongly detected when the disease worsened.

また、上記により得られた15種類の肺サルコイドーシス特異的His−tag標識自己抗原蛋白をNi−NT(図4)SEREX法によって検出した肺サルコイドーシス特異的自己抗体の認識する自己抗原蛋白とその発現頻度を調べた。A標識ELISAプレートに添加し4度で一晩インキュベートし固定した。これによりHis−tag標識蛋白をNi−NTAに結合させることができ、これにより得られたELISAプレートを用いて症例血清およびBAL液を用いて自己抗原蛋白と結合する自己抗体濃度を検出した。His−tag標識自己抗原蛋白を吸着させたプレートによる血清およびBAL液によるELISA解析では、病勢が悪化した場合に抗体価が高く検出される傾向が認められた。本発明のELISA解析は、肺サルコイドーシスおよび眼サルコイドーシスの病勢フォローに有用な指標であった。   Furthermore, the 15 types of pulmonary sarcoidosis-specific His-tag labeled autoantigen proteins obtained as described above were detected by the Ni-NT (FIG. 4) SEREX method, and the self-antigen protein recognized by the pulmonary sarcoidosis-specific autoantibodies and their expression frequencies. I investigated. It was added to an A-labeled ELISA plate and incubated overnight at 4 degrees for fixation. As a result, the His-tag labeled protein could be bound to Ni-NTA, and the concentration of autoantibodies bound to the self-antigen protein was detected using the case serum and the BAL fluid using the ELISA plate thus obtained. In the ELISA analysis using serum and BAL fluid with a plate adsorbed with a His-tag labeled autoantigen protein, a tendency was found that the antibody titer was detected higher when the disease state worsened. The ELISA analysis of the present invention was a useful index for follow-up of pulmonary sarcoidosis and ocular sarcoidosis.

T細胞Vβ鎖サブファミリーに対するPCR法に用いたプライマーを示す図。The figure which shows the primer used for PCR method with respect to T cell V (beta) chain | strand subfamily. 肺サルコイドーシスにおけるT細胞Vβ鎖サブファミリーの解析の例。Example of analysis of T cell Vβ chain subfamily in pulmonary sarcoidosis. SEREX法の概略図を示す図である。It is a figure which shows the schematic of a SEREX method. SEREX法によって検出した肺サルコイドーシス特異的自己抗体の認識する自己抗原蛋白とその発現頻度を示す図。The figure which shows the self-antigen protein which the lung sarcoidosis specific autoantibody recognized by the SEREX method recognizes, and its expression frequency. SEREX法によって検出した肺サルコイドーシス特異的自己抗体の認識する肺胞と眼共通自己抗原蛋白とその発現頻度を示す図。The figure which shows the alveoli recognized by the pulmonary sarcoidosis specific autoantibody detected by the SEREX method, an ocular common autoantigen protein, and its expression frequency. 発現頻度の高い3種類の自己抗体の発現状況を示す図。The figure which shows the expression condition of three types of autoantibodies with high expression frequency. 肺サルコイドーシス自己抗体認識抗原遺伝子を増幅するために用いたPCRプライマーを示す図。The figure which shows the PCR primer used in order to amplify the pulmonary sarcoidosis autoantibody recognition antigen gene. RT−PCR法によって3種類の自己抗原蛋白のII型肺胞上皮癌培養株(A549)および単球系培養株(THP−1)における発現を解析した図。 1:retinal short−chain dehydrogenase/reductase (ret SDR2)、 2:1−phosphatidylinositol−4−phosphate 5 kinase isoform c、 3:poly a binding protein 1、A:A549cell line、T:THP−1cell lineThe figure which analyzed the expression in the type II alveolar epithelial cancer culture strain (A549) and monocyte culture strain (THP-1) of three types of autoantigen proteins by RT-PCR method. 1: retinal short-chain dehydrogenase / reductase (ret SDR2), 2: 1-phosphatidylinosideol-4-phosphate 5 kinase isoform cell, TH: poly a binding clot

Claims (4)

抗poly a binding protein 1抗体を含有してなる肺サルコイドーシスの検出マーカー。 A marker for detecting pulmonary sarcoidosis comprising an anti-poly a binding protein 1 antibody . poly a
binding protein 1抗原タンパクを基材に吸着させてなる肺サルコイドーシスの検出キット。
poly a
A detection kit for pulmonary sarcoidosis comprising binding protein 1 antigen protein adsorbed on a base material.
前記抗原タンパクは標識が付されている請求項2記載の肺サルコイドーシスの検出キット。 The pulmonary sarcoidosis detection kit according to claim 2, wherein the antigen protein is labeled. 前記標識としてHis−tag標識が付されている請求項3記載の肺サルコイドーシスの検出キット。 The kit for detecting pulmonary sarcoidosis according to claim 3, wherein a His-tag label is attached as the label.
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