JP2011117781A - Immunoassay method for complex of clathrin heavy chain and autoantibody of the same, kit used for the same, and cancer determination method using the same - Google Patents
Immunoassay method for complex of clathrin heavy chain and autoantibody of the same, kit used for the same, and cancer determination method using the same Download PDFInfo
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Abstract
Description
本発明は、クラスリン重鎖とその自己抗体との複合体を測定するための免疫測定方法及び免疫測定用キットに関するものである。クラスリン重鎖とその自己抗体との複合体は、特に担癌患者の血中に高値に出現するものであり、したがって、本発明は、クラスリン重鎖とその自己抗体との複合体を単に測定する方法を提供するだけでなく、癌の判定にも利用することができる。 The present invention relates to an immunoassay method and an immunoassay kit for measuring a complex of a clathrin heavy chain and its autoantibody. The complex of clathrin heavy chain and its autoantibody is particularly high in the blood of cancer-bearing patients, so the present invention simply refers to the complex of clathrin heavy chain and its autoantibody. In addition to providing a method of measurement, it can also be used to determine cancer.
クラスリン (clathrin) は、細胞外マトリクスの分子がエンドサイトーシスにより取り込まれる際に形成される、エンドソーム外側を形作る骨格となるタンパク質である。クラスリン分子は三脚巴構造 (triskelion) を取り、エンドソーム形成時は、複数のクラスリンが重合して格子を形成する。クラスリン分子は三つの足を持ち、三脚巴構造を取り、それぞれの足はクラスリン重鎖とクラスリン軽鎖からなる。クラスリン重鎖は１６７５アミノ酸残基よりなり、分子量は約１９２ｋＤａである。クラスリン軽鎖の分子量は約２５−２９ｋＤａである（非特許文献１）。
他方、アガロース２次元電気泳動に２Ｄ−ＤＩＧＥ法（two-dimensional fluorescence difference gel electrophrosis）を適用した改良アガロース２次元電気泳動法（非特許文献２）により、原発性肝細胞癌の癌部および周辺の非癌部組織の蛋白発現量の比較をプロテオーム解析を行ない、クラスリン重鎖が癌部に多く発現されることが明らかになっている（非特許文献３）
Clathrin is a protein that forms a framework that forms the outside of endosomes, which is formed when molecules in the extracellular matrix are taken up by endocytosis. The clathrin molecule has a triskelion structure, and during endosome formation, multiple clathrins are polymerized to form a lattice. The clathrin molecule has three legs and has a tripodal structure, each leg consisting of a clathrin heavy chain and a clathrin light chain. The clathrin heavy chain consists of 1675 amino acid residues and has a molecular weight of about 192 kDa. The molecular weight of the clathrin light chain is about 25-29 kDa (Non-patent Document 1).
On the other hand, by the improved agarose two-dimensional electrophoresis method (Non-patent Document 2) applying 2D-DIGE (two-dimensional fluorescence difference gel electrophrosis) to agarose two-dimensional electrophoresis, the cancerous part of the primary hepatocellular carcinoma and the surrounding area Proteome analysis is performed to compare protein expression levels in non-cancerous tissues, and it has been clarified that clathrin heavy chains are often expressed in cancerous parts (Non-patent Document 3).
本発明者らは、癌が疑われる患者または癌患者由来の組織検体中に存在する、クラスリン重鎖の発現レベルを測定して、それらの組織の癌部と非癌部の判別することができることを見出し、これに対し特許出願した（特願２００８年２６４７９４号）。本発明者らは、さらに血液検体中のクラスリン重鎖の存在について研究を続けたところ、驚くべきことに、血液検体中にクラスリン重鎖とその自己抗体との複合体が存在する場合があり、癌患者の場合、その複合体の量が多いことを見出した。したがって、本発明の目的は、癌判定に応用することのできる、クラスリン重鎖とその自己抗体との複合体の測定方法を提供することである。 The present inventors can measure the expression level of clathrin heavy chain present in a tissue specimen derived from a patient suspected of cancer or a cancer patient to discriminate between a cancerous part and a non-cancerous part of those tissues. We found out what we could do and filed a patent application for this (Japanese Patent Application No. 264794). The present inventors further continued research on the presence of clathrin heavy chain in a blood sample. Surprisingly, a complex of clathrin heavy chain and its autoantibody may exist in a blood sample. Yes, in the case of cancer patients, the amount of the complex was found to be large. Therefore, an object of the present invention is to provide a method for measuring a complex of a clathrin heavy chain and its autoantibody that can be applied to cancer determination.
本発明者らは、検体中のクラスリン重鎖とその自己抗体との複合体を免疫測定するにより、該複合体を測定することができ、それによって癌判定が可能になることを見出し、本発明を完成させた。
従って、本発明は、検体中のクラスリン重鎖とその自己抗体との複合体を免疫測定することを特徴とする、検体中のクラスリン重鎖とその自己抗体との複合体の免疫測定方法に関する。
更に、本発明は、クラスリン重鎖に対する試薬抗体およびその自己抗体に対する結合可能物質を含む、クラスリン重鎖とその自己抗体との複合体の免疫測定用キットに関する。
更に、本発明は、クラスリン重鎖とその自己抗体との複合体を測定することにより、癌であることを判定する、癌判定方法に関する。
The present inventors have found that the complex can be measured by immunoassaying the complex of clathrin heavy chain and its autoantibody in a specimen, thereby enabling cancer determination. Completed the invention.
Accordingly, the present invention provides a method for immunoassay of a complex of a clathrin heavy chain and its autoantibody in a sample, characterized by immunoassay of the complex of the clathrin heavy chain and its autoantibody in the sample. About.
Furthermore, the present invention relates to a kit for immunoassay of a complex of a clathrin heavy chain and its autoantibody comprising a reagent antibody against the clathrin heavy chain and a substance capable of binding to the autoantibody.
Furthermore, the present invention relates to a cancer determination method for determining cancer by measuring a complex of a clathrin heavy chain and its autoantibody.
本発明においては、検体中、特に、血液由来検体中に存在するクラスリン重鎖とその自己抗体との複合体を簡単に測定でき、原発性肝細胞癌などの癌患者の判定に有効である。 In the present invention, a complex of a clathrin heavy chain and its autoantibody can be easily measured in a specimen, particularly in a blood-derived specimen, and is effective for determination of cancer patients such as primary hepatocellular carcinoma. .
本発明のクラスリン重鎖とその自己抗体との複合体の免疫測定方法は、一般的な蛋白質とその抗体との免疫複合体の免疫測定方法で知られている公知の方法をそのまま、応用できる。
本発明の免疫測定方法においては、例えば、検体中のクラスリン重鎖とその自己抗体との複合体に、クラスリン重鎖に対する試薬抗体およびその自己抗体に対する結合可能物質を作用させ、得られる複合体と試薬抗体と結合可能物質との免疫複合物を測定することによりその複合体を測定することができる。
As a method for immunoassay of a complex of clathrin heavy chain and its autoantibody according to the present invention, a known method known as an immunoassay method for immunocomplex of general protein and its antibody can be applied as it is. .
In the immunoassay method of the present invention, for example, a complex obtained by allowing a reagent antibody against a clathrin heavy chain and a substance capable of binding to the autoantibody to act on a complex of a clathrin heavy chain and its autoantibody in a specimen. The complex can be measured by measuring the immune complex of the body, the reagent antibody, and the bindable substance.
本発明において、検体とは、生体由来の試料が好適で、特に、血液由来検体が好適であり、血液検体としては、全血、血漿、血清を例示できる。 In the present invention, the sample is preferably a biological sample, particularly a blood-derived sample, and examples of the blood sample include whole blood, plasma, and serum.
本発明の免疫測定方法の測定対象は、クラスリン重鎖とその自己抗体との複合体であり、このクラスリン重鎖は、細胞膜および細胞内小器官の表面で細胞内輸送の受容体や様々な高分子物質の貪食に関与する蛋白質であるクラスリンの構成成分のひとつである。クラスリン重鎖は、ＣＨＣとも標記され、Ｓｗｉｓｓ−ＰｒｏｔｅｎｔｒｙＮｏ，Ｑ００１６０でコードされる分子量１９２ｋＤａ、１６７５アミノ酸で構成される蛋白質であり、Ｈｃ；ＣＨＣ；ＣＨＣ１７；ＣＬＨ−１７；ＣＬＴＣＬ２；ＫＩＡＡ００３４；ＣＬＴＣ等の略称を持つ。 The measurement target of the immunoassay method of the present invention is a complex of clathrin heavy chain and its autoantibody, and this clathrin heavy chain is a receptor for intracellular transport and various kinds of cells on the surface of cell membranes and organelles. It is one of the components of clathrin, a protein involved in the phagocytosis of various high molecular substances. The clathrin heavy chain is a protein composed of CHC, CHC17; CLH-17; CLTCL2; KIAA0034; CLTC, and the like. Has an abbreviation.
本発明において、クラスリン重鎖とその自己抗体との複合体とは、クラスリン重鎖とクラスリン重鎖に対する自己抗体との免疫複合体を意味し、本明細書では単に複合体と記載することもある。
本発明において、自己抗体とは、自己の身体に存在する物質に対して自己の身体で産生される抗体であって、自己の身体に存在する物質がクラスリン重鎖であり、そのクラスリン重鎖に対する抗体をいう。
本発明において、クラスリン重鎖に対する試薬抗体とは、試薬として用いるクラスリン重鎖と特異的に結合する抗体をいい、本明細書では単に試薬抗体と記載することもある。その試薬抗体は、その産生動物種としてヒト、マウス、ラット、ウサギ、ヤギ、ウマ等があり、それぞれに所定範囲の免疫グロブリンがある。その試薬抗体は、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＥ、ＩｇＤのいずれでもよい。また、試薬抗体は、モノクローナル抗体、ポリクローナル抗体、及びこれらの断片（抗原と結合能を有するもので例えば、Ｈ鎖、Ｌ鎖、Ｆａｂ、Ｆ（ａｂ’）２等）のいずれでもよい。このような試薬抗体は、クラスリン重鎖全長蛋白質またはその断片ペプチドを抗原として、上記した産生動物種に免疫して、その免疫動物から抗血清として得ることができ、また、免疫動物からの脾細胞とミエローマ細胞とを融合して、融合細胞から、クラスリン重鎖に対する抗体を産生する融合細胞をスクリーニングして、得られるハイブリドーマからモノクローナル抗体として得ることもできる。また、クラスリン重鎖に対する試薬抗体は、抗クラスリン重鎖抗体として市販されており、それらの市販品を使用することもできる。
In the present invention, the complex of clathrin heavy chain and its autoantibody means an immune complex of clathrin heavy chain and autoantibody against clathrin heavy chain, and is simply described as a complex in this specification. Sometimes.
In the present invention, an autoantibody is an antibody produced in the own body against a substance present in the own body, and the substance present in the own body is a clathrin heavy chain, An antibody against the chain.
In the present invention, a reagent antibody against a clathrin heavy chain refers to an antibody that specifically binds to a clathrin heavy chain used as a reagent, and may be simply referred to as a reagent antibody in this specification. The reagent antibodies include humans, mice, rats, rabbits, goats, horses, and the like as production animal species, and each has a predetermined range of immunoglobulins. The reagent antibody may be any of IgG, IgM, IgA, IgE, and IgD. The reagent antibody may be any of a monoclonal antibody, a polyclonal antibody, and a fragment thereof (having an antigen-binding ability, such as H chain, L chain, Fab, F (ab ′) 2, etc.). Such a reagent antibody can be obtained as an antiserum from an immunized animal by immunizing the above-mentioned production animal species using a clathrin heavy chain full-length protein or a fragment peptide thereof as an antigen, and a spleen from the immunized animal. It is also possible to obtain a monoclonal antibody from the resulting hybridoma by fusing a cell and a myeloma cell, screening a fused cell that produces an antibody against clathrin heavy chain from the fused cell. Moreover, the reagent antibody with respect to a clathrin heavy chain is marketed as an anti- clathrin heavy chain antibody, and those commercial products can also be used.
本発明において、その自己抗体に対する結合可能物質とは、クラスリン重鎖に対する自己抗体と結合可能な物質であれば特に限定しないが、本明細書では単に結合可能物質と記載することがある。この様な結合可能物質としては、抗ＩｇＧ抗体、プロテインＡ、プロテインＧ、試薬としてのクラスリン重鎖抗原を用いることができ、そのうち、抗ＩｇＧ抗体が好ましい。
試薬としてのクラスリン重鎖抗原を用いる場合、クラスリン重鎖に対する自己抗体と抗原抗体反応しうる抗原であれば特に限定しないが、クラスリン重鎖全長蛋白質、クラスリン重鎖全長蛋白質の変異体であって該蛋白質と同様のクラスリン重鎖に対する自己抗体と抗原抗体反応しうる機能を有し且つ該アミノ酸配列と９０％以上の相同性を有する蛋白質もしくはクラスリン重鎖全長蛋白質のアミノ酸配列において１個から数個のアミノ酸残基が欠失、置換もしくは付加したアミノ酸配列を有する蛋白質である変異体、クラスリン重鎖の断片ペプチドであってクラスリン重鎖の自己抗体と抗原抗体反応しうるペプチドを例示できる。クラスリン重鎖全長蛋白質は、シグマ社より入手可能であるが、全アミノ酸配列が既知であるので、クラスリン重鎖全長蛋白質やその変異体は、遺伝子組換え技術によっても合成できる。本発明においてクラスリン重鎖の断片ペプチドを用いるときは、クラスリン重鎖全長蛋白質を酵素分解等によって各種のペプチド断片に切断して作成してもよいし、市販の自動ペプチド合成装置を用いても容易に作成することができる。また、標的のクラスリン重鎖の断片ペプチドを遺伝子組み換え技術によっても作成することができる。
そのようにして得られたクラスリン重鎖全長蛋白質の変異体や断片ペプチドを、クラスリン重鎖に対する自己抗体と反応させ抗原抗体反応をするものを選択して試薬としてのクラスリン重鎖抗原として用いることができる。本発明においては、上記した各ペプチド断片の全体のほか、その一部も使用できるし、それらの混合物も使用でき、これらも試薬としてのクラスリン重鎖抗原に包含される。
In the present invention, the substance capable of binding to the autoantibody is not particularly limited as long as it is a substance capable of binding to the autoantibody against the clathrin heavy chain, but may be simply described as a substance capable of binding in the present specification. As such a substance capable of binding, anti-IgG antibody, protein A, protein G, and clathrin heavy chain antigen as a reagent can be used, and of these, anti-IgG antibody is preferable.
When using a clathrin heavy chain antigen as a reagent, any antigen capable of antigen-antibody reaction with an autoantibody against clathrin heavy chain is not particularly limited, but a clathrin heavy chain full length protein, a clathrin heavy chain full length protein variant In the amino acid sequence of a protein having a function capable of antigen-antibody reaction with an autoantibody against a clathrin heavy chain similar to that of the protein or having a homology of 90% or more with the amino acid sequence or a full-length clathrin heavy chain protein A variant that is a protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted, or added, a fragment peptide of a clathrin heavy chain that can react with an antigen antibody with a clathrin heavy chain autoantibody Peptides can be exemplified. The clathrin heavy chain full-length protein is available from Sigma, but since the entire amino acid sequence is known, the clathrin heavy chain full-length protein and its mutants can also be synthesized by gene recombination techniques. When a fragment peptide of clathrin heavy chain is used in the present invention, the clathrin heavy chain full-length protein may be prepared by cleaving into various peptide fragments by enzymatic degradation or the like, or using a commercially available automatic peptide synthesizer. Can also be created easily. In addition, a fragment peptide of the target clathrin heavy chain can be prepared by a gene recombination technique.
The so-obtained clathrin heavy chain full-length protein mutant or fragment peptide is reacted with an autoantibody against clathrin heavy chain to select an antigen-antibody reaction and used as a clathrin heavy chain antigen as a reagent. Can be used. In the present invention, in addition to the whole peptide fragments described above, a part of them can be used, or a mixture thereof can be used, and these are also included in the clathrin heavy chain antigen as a reagent.
本発明においては、検体中のクラスリン重鎖とその自己抗体との複合体に、クラスリン重鎖に対する試薬抗体およびその自己抗体に対する結合可能物質を作用させると、複合体と試薬抗体と結合可能物質との免疫複合物が生成する。その免疫複合物を測定するには、クラスリン重鎖に対する試薬抗体（試薬抗体）およびその自己抗体に対する結合可能物質（結合可能物質）のいずれかを標識成分で標識し、生成する免疫複合物中の標識成分を測定することによって、免疫複合物を測定するのが好ましい。
標識成分としては、酵素、放射性物質、蛍光物質、化学発光物質等常用される標識成分を使用することができるが、酵素や放射性物質が好ましい。
標識するための酵素としては、西洋ワサビペルオキシダーゼ(ＨＲＰ)、ウシ小腸アルカリフォスファターゼ、β−ガラクトシダーゼ、ウレアーゼ、グルコースオキシダーゼ等の酵素免疫分析法（ＥＩＡ）に常用される酵素が適宜使用され、これらの酵素に適合しＥＩＡで常用される発色基質が適宜使用される。発色基質としては、例えばＨＲＰの場合は、３，３′，５，５′−テトラメチルベンジジン（ＴＭＢＺ）、ＴＭＢＺ・ＨＣｌ、ＴＭＢＺ・ＰＳ、ＡＢＴＳ、ｏ−フェニレンジアミン、ｐ−ヒドロキシフェニル酢酸等が使用され、アルカリフォスファターゼの場合は、ｐ−ニトロフェニルフォスフェート、４−メチルウンベリフェリルフォスフェート等が使用され、β−ガラクトシダーゼの場合は、ｏ−ニトロフェニル−β−Ｄ−ガラクトピラノシド、４−メチルウンベリフェリルβ−Ｄ−ガラクトピラノシド等が使用される。
標識するための放射性物質としては放射性ヨウ素原子等を、蛍光物質としてはＦＩＴＣやローダミン等を、化学発光物質としてはルミノール等を例示することができる。
In the present invention, when a reagent antibody against clathrin heavy chain and a substance capable of binding to autoantibody are allowed to act on a complex of clathrin heavy chain and its autoantibody in a sample, the complex and reagent antibody can be bound. An immune complex with the substance is produced. To measure the immune complex, either the reagent antibody against the clathrin heavy chain (reagent antibody) or the substance capable of binding to the autoantibody (binding substance) is labeled with a labeling component, It is preferred to measure the immune complex by measuring the labeled component.
As the labeling component, commonly used labeling components such as enzymes, radioactive substances, fluorescent substances, and chemiluminescent substances can be used, but enzymes and radioactive substances are preferred.
As the enzyme for labeling, enzymes commonly used in enzyme immunoassay (EIA) such as horseradish peroxidase (HRP), bovine small intestine alkaline phosphatase, β-galactosidase, urease, glucose oxidase and the like are appropriately used. A chromogenic substrate that is suitable for EIA and commonly used in EIA is appropriately used. As the chromogenic substrate, for example, in the case of HRP, 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), TMBZ · HCl, TMBZ · PS, ABTS, o-phenylenediamine, p-hydroxyphenylacetic acid, etc. In the case of alkaline phosphatase, p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, etc. are used, and in the case of β-galactosidase, o-nitrophenyl-β-D-galactopyranoside, 4-Methylumbelliferyl β-D-galactopyranoside and the like are used.
Examples of radioactive materials for labeling include radioactive iodine atoms, examples of fluorescent materials include FITC and rhodamine, and examples of chemiluminescent materials include luminol.
本発明において標識成分を用いる場合、例えば、検体中のクラスリン重鎖とその自己抗体との複合体に、水不溶性担体に結合しているクラスリン重鎖に対する試薬抗体を作用させ、次いで、標識成分で標識されたその自己抗体に対する結合可能物質を作用させ、複合体と試薬抗体と結合可能物質との免疫複合物を生成させ、その免疫複合物に結合している標識成分を測定することによりその複合体を測定することが好ましい。また、検体中のクラスリン重鎖とその自己抗体との複合体に、その自己抗体に対する結合可能物質が結合した水不溶性担体を作用させ、次いで、標識成分で標識されたクラスリン重鎖に対する試薬抗体を作用させ、複合体と結合可能物質と試薬抗体との免疫複合物を生成させ、その免疫複合物に結合している標識成分を測定することによりその複合体を測定することもできる。 When a labeling component is used in the present invention, for example, a reagent antibody against a clathrin heavy chain bound to a water-insoluble carrier is allowed to act on a complex of a clathrin heavy chain and its autoantibody in a sample, and then labeled By reacting a substance capable of binding to the autoantibody labeled with the component, generating an immune complex of the complex, the reagent antibody and the substance capable of binding, and measuring the labeled component bound to the immune complex. It is preferable to measure the complex. In addition, a reagent for clathrin heavy chain labeled with a labeling component is allowed to act on a complex of clathrin heavy chain in the sample and its autoantibody with a water-insoluble carrier bound with a substance capable of binding to the autoantibody. The complex can also be measured by causing an antibody to act, generating an immune complex of the complex, a binding substance and a reagent antibody, and measuring the label component bound to the immune complex.
水不溶化担体の調製は、蛋白質を固相面に結合する既知の方法を用いて容易に行うことができる。例えば、固相化担体としては、通常、ビーズ、マイクロプレート、チューブ等が用いられる。これらの固相面にクラスリン重鎖に対する試薬抗体を結合する方法としては、物理吸着、化学結合等既知の固定化技術が適宜利用できる。
このようにして固相化したクラスリン重鎖に対する試薬抗体に、クラスリン重鎖とその自己抗体との複合体とを含む検体とを接触させると、複合体中のクラスリン重鎖部分と試薬抗体とが結合する。さらに、その結合物に対し、標識成分で標識されたその自己抗体に対する結合可能物質（例えば標識抗ＩｇＧ抗体）を作用させると複合体と試薬抗体と結合可能物質との免疫複合物が生成する。その結果、生成する免疫複合物中の標識成分を測定することにより、検体中のクラスリン重鎖とその自己抗体との複合体を測定することができる。
上記と同様にして、固相化担体にクラスリン重鎖の自己抗体に対する結合可能物質を結合させて、固相化した結合可能物質に、クラスリン重鎖とその自己抗体との複合体とを含む検体とを接触させて、複合体中の自己抗体部分と結合可能物質とを結合させ、さらに、その結合物に対し、標識成分で標識されたクラスリン重鎖に対する試薬抗体（例えば、抗クラスリン重鎖抗体）を作用させて、複合体と結合可能物質と試薬抗体との免疫複合物を生成させて、同様にして、検体中のクラスリン重鎖とその自己抗体との複合体を測定することもできる。
The water-insolubilized carrier can be easily prepared using a known method for binding a protein to a solid phase surface. For example, as the solid-phase support, beads, microplates, tubes and the like are usually used. As a method for binding a reagent antibody against clathrin heavy chain to these solid phase surfaces, known immobilization techniques such as physical adsorption and chemical bonding can be used as appropriate.
When the reagent antibody against the clathrin heavy chain thus immobilized is brought into contact with a specimen containing the complex of clathrin heavy chain and its autoantibody, the clathrin heavy chain portion in the complex and the reagent The antibody binds. Further, when a substance capable of binding to the autoantibody labeled with a labeling component (for example, a labeled anti-IgG antibody) is allowed to act on the conjugate, an immunocomplex of the complex, the reagent antibody and the substance capable of binding is produced. As a result, the complex of the clathrin heavy chain and its autoantibody in the specimen can be measured by measuring the labeled component in the generated immune complex.
In the same manner as described above, a substance capable of binding to the autoantibody of clathrin heavy chain is bound to the immobilized carrier, and the complex of clathrin heavy chain and the autoantibody is bound to the immobilized substance capable of binding. A reagent antibody against the clathrin heavy chain labeled with a labeling component (for example, an anti-class) Phospho-heavy chain antibody) is acted on to generate an immunocomplex of the complex, a binding substance and a reagent antibody, and in the same way, the complex of the clathrin heavy chain and its autoantibody in the sample is measured. You can also
本発明の免疫測定方法の典型的な例を以下に示す。
プレートに抗クラスリン重鎖抗体を加え、低温例えば４℃で静置して感作し、その後、ＰＢＳ等の洗浄液で洗浄する。ついで、そのプレートをＢＳＡでコーティングし、抗クラスリン重鎖抗体ＥＬＩＳＡプレートを作成する。希釈した検体を抗クラスリン重鎖抗体ＥＬＩＳＡプレートに加え、加温例えば３７℃で静置し、次いでＰＢＳ等の洗浄液で洗浄をする。得られるプレートのウェルにＨＲＰ標識された抗ヒトＩｇＧ抗体を加え、加温例えば３７℃で静置する。ついで、ウェルをＰＢＳ等の洗浄液で洗浄した後、ＴＭＢＺを加え、例えば室温で静置した後、反応停止剤として１Ｎ硫酸を加える。吸光度はマイクロプレートリーダー（ＢｉｏＲａｄ社製）を用いて、波長４５０ｎｍにて吸光度を測定する。吸光度の値とあらかじめ作成しておいた検量線から、クラスリン重鎖とその自己抗体との複合体の値を求める。
A typical example of the immunoassay method of the present invention is shown below.
Anti-clathrin heavy chain antibody is added to the plate, left to stand at a low temperature, for example, 4 ° C. for sensitization, and then washed with a washing solution such as PBS. The plate is then coated with BSA to create an anti-clathrin heavy chain antibody ELISA plate. The diluted specimen is added to the anti-clathrin heavy chain antibody ELISA plate, left to stand at a warm temperature, for example, 37 ° C., and then washed with a washing solution such as PBS. An HRP-labeled anti-human IgG antibody is added to the well of the resulting plate, and the plate is allowed to stand at a warm temperature, for example, 37 ° C. Next, the well is washed with a washing solution such as PBS, and then TMBZ is added. For example, after standing at room temperature, 1N sulfuric acid is added as a reaction terminator. Absorbance is measured at a wavelength of 450 nm using a microplate reader (BioRad). From the absorbance value and a calibration curve prepared in advance, the value of the complex of clathrin heavy chain and its autoantibody is determined.
本発明の測定方法は、クラスリン重鎖に対する試薬抗体およびその自己抗体に対する結合可能物質を含む、クラスリン重鎖とその自己抗体との複合体の免疫測定用キットにより実施することができる。そのためのクラスリン重鎖に対する試薬抗体、その自己抗体に対する結合可能物質は、本発明の測定方法で説明したとおりである。すなわち、例えば、クラスリン重鎖に対する試薬抗体およびその自己抗体に対する結合可能物質のいずれかを水不溶性担体に結合させた形態で、他のいずれかを標識成分で標識した形態で、キットの試薬成分とすることができる。その他の試薬成分として、界面活性剤、緩衝剤等の免疫測定法で常用されるものを適宜、加えてもよい。 The measurement method of the present invention can be carried out with a kit for immunoassay of a complex of a clathrin heavy chain and its autoantibody, which contains a reagent antibody against the clathrin heavy chain and a substance capable of binding to the autoantibody. For this purpose, the reagent antibody against the clathrin heavy chain and the substance capable of binding to the autoantibody are as described in the measurement method of the present invention. That is, for example, the reagent component of the kit in a form in which either a reagent antibody to clathrin heavy chain or a substance capable of binding to its autoantibody is bound to a water-insoluble carrier, and the other is labeled with a labeling component. It can be. As other reagent components, those commonly used in immunoassays such as surfactants and buffers may be added as appropriate.
本発明においては、クラスリン重鎖とその自己抗体との複合体を測定することにより、癌判定をすることができる。
また、一般にクラスリン重鎖とその自己抗体との複合体の量が多いと、原発性肝細胞癌（原発性肝細胞癌、原発性胆管細胞癌など）、転移性肝癌等の肝癌、膀胱癌、乳癌、肺癌、卵巣癌、前立腺癌、甲状腺癌、皮膚癌などの癌が疑われ、本発明によりクラスリン重鎖に対する自己抗体を測定することは、患者の癌疾患の判別に有効である。本発明においては、原発性肝細胞癌の判別に有効であり、例えば、健常人と、初発原発性肝細胞癌患者や再発原発性肝細胞癌患者との判別に有効である。
In the present invention, cancer can be determined by measuring a complex of clathrin heavy chain and its autoantibody.
In general, when the amount of the complex of clathrin heavy chain and its autoantibody is large, primary hepatocellular carcinoma (primary hepatocellular carcinoma, primary cholangiocellular carcinoma etc.), liver cancer such as metastatic liver cancer, bladder cancer Cancers such as breast cancer, lung cancer, ovarian cancer, prostate cancer, thyroid cancer, and skin cancer are suspected, and measurement of autoantibodies against clathrin heavy chain according to the present invention is effective for discriminating cancer diseases in patients. In the present invention, it is effective for discriminating primary hepatocellular carcinoma. For example, it is effective for discriminating between healthy individuals and patients with primary primary hepatocellular carcinoma or patients with recurrent primary hepatocellular carcinoma.
以下、本発明を実施例により更に詳細に説明するが、本発明は、これら実施例に何ら制限されるものではない。
実施例１
クラスリン重鎖とその自己抗体との複合体の測定
健常人、初発原発性肝細胞癌患者、及び再発原発性肝細胞癌患者から採取した血清検体について、クラスリン重鎖とその自己抗体との複合体の測定を、以下に具体的に説明するようにして行なった。
EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not restrict | limited to these Examples at all.
Example 1
Measurement of the complex of clathrin heavy chain and its autoantibodies Serum samples collected from healthy subjects, patients with primary hepatocellular carcinoma, and patients with recurrent primary hepatocellular carcinoma The measurement of the complex was performed as specifically described below.
１．方法
（１）抗クラスリン重鎖抗体ＥＬＩＳＡプレートの作成
ＥＬＩＳＡプレート（Ｎｕｎｃ社製，Ｍａｘｉｓｏｒｐ）に抗クラスリン重鎖抗体（ＢＤ Ｂｉｏｓｉｅｎｓｅｓ Ｐｈａｒｍｉｎｇｅｎ社製，５μｇ／ｍＬ，１００μＬ／ｗｅｌｌ）を１晩４℃静置して感作し、その後、０．０５％Ｔｗｅｅｎ２０を含むＰＢＳ（２００μＬ／ｗｅｌｌ）で３回洗浄を行った。ついで、１．５％ＢＳＡ、１０％サッカロースを含むＰＢＳ（２００μＬ／ｗｅｌｌ）で１晩コーティングし、抗クラスリン重鎖抗体ＥＬＩＳＡプレートを作成した。
1. Method (1) Preparation of anti-clathrin heavy chain antibody ELISA plate An anti-clathrin heavy chain antibody (BD Biosciences Pharmingen, 5 μg / mL, 100 μL / well) is applied to an ELISA plate (Nunc, Maxisorp) overnight at 4 ° C. The mixture was allowed to stand for sensitization, and then washed three times with PBS containing 0.05% Tween 20 (200 μL / well). Subsequently, it was coated overnight with PBS (200 μL / well) containing 1.5% BSA and 10% saccharose to prepare an anti-clathrin heavy chain antibody ELISA plate.
（２）クラスリンとその自己抗体との複合体の測定
検出抗体としてＨＲＰ標識された抗ヒトＩｇＧ抗体（Ｚｙｍｅｄ社製）を、０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳにて４０００倍に希釈したものを用いた。サンプル血清はＰＢＳにて１００倍に希釈した。その希釈したサンプルを抗クラスリン重鎖抗体ＥＬＩＳＡプレートに１００μＬ／ｗｅｌｌずつ加え、１時間３７℃で静置し、その後、０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳ（２００μＬ／ｗｅｌｌ）で３回洗浄を行った。得られるプレートのウェルに希釈したＨＲＰ標識された抗ヒトＩｇＧ抗体を１００μＬ／ｗｅｌｌずつ加え、３０分間３７℃で静置した。ついで、０．０５％Ｔｗｅｅｎ２０を含むＰＢＳ（２００μＬ／ｗｅｌｌ）で３回洗浄した後、ＴＭＢＺを１００μＬ／ｗｅｌｌずつ加え、１０分間室温で静置の後、反応停止剤として１００μＬ／ｗｅｌｌの１Ｎ硫酸を加えた。吸光度はマイクロプレートリーダー（ＢｉｏＲａｄ社製）を用いて、波長４５０ｎｍにて測定を行った。
なお、検体は健常人１６例、初発原発性肝細胞癌患者検体１６症例、再発原発性肝細胞癌患者１６例を用いた。
(2) Measurement of a complex of clathrin and its autoantibody A HRP-labeled anti-human IgG antibody (manufactured by Zymed) as a detection antibody diluted 4000 times with PBS containing 0.05% Tween 20 Using. Sample serum was diluted 100 times with PBS. The diluted sample was added to an anti-clathrin heavy chain antibody ELISA plate at 100 μL / well, allowed to stand at 37 ° C. for 1 hour, and then washed three times with PBS containing 0.05% Tween 20 (200 μL / well). It was. 100 μL / well of diluted HRP-labeled anti-human IgG antibody was added to each well of the resulting plate, and allowed to stand at 37 ° C. for 30 minutes. Next, after washing three times with PBS containing 0.05% Tween 20 (200 μL / well), 100 μL / well of TMBZ was added, and the mixture was allowed to stand at room temperature for 10 minutes. Then, 100 μL / well of 1N sulfuric acid was added as a reaction stopper. added. Absorbance was measured at a wavelength of 450 nm using a microplate reader (BioRad).
The specimens used were 16 healthy subjects, 16 primary hepatocellular carcinoma patient specimens, and 16 recurrent primary hepatocellular carcinoma patients.
２．結果
クラスリン重鎖とその自己抗体との複合体を測定した結果を用いた結果を、図１に示す。有意差検定はＫａｌｅｉｄａＧｒａｐｈ４．０を用い、Ｗｉｌｃｏｘｏｎの２標本検定にて統計処理した。
図１に示すように、健常人群と比較し、初発原発性肝細胞癌患者検体群、再発原発性肝細胞癌患者検体群のクラスリン重鎖とその自己抗体との複合体は、明らかな有意差を認めた。
2. Results FIG. 1 shows the results obtained by measuring the complex of clathrin heavy chain and its autoantibody. For the significant difference test, KaleidaGraph 4.0 was used, and statistical processing was performed by Wilcoxon two-sample test.
As shown in FIG. 1, the complex of clathrin heavy chain and its autoantibody in the primary primary hepatocellular carcinoma patient sample group and the recurrent primary hepatocellular carcinoma patient sample group is clearly significant compared to the healthy group. There was a difference.
以上に詳細に説明したように、血液由来検体などの検体中のクラスリン重鎖とその自己抗体との複合体に、クラスリン重鎖に対する試薬抗体およびその自己抗体に対する結合可能物質を作用させ、得られる複合体と試薬抗体と結合可能物質との免疫複合物を測定することによりその複合体を測定することができ、それによって、原発性肝細胞癌などの癌判定が可能である。 As explained in detail above, a reagent antibody against a clathrin heavy chain and a substance capable of binding to the autoantibody are allowed to act on a complex of a clathrin heavy chain and its autoantibody in a specimen such as a blood-derived specimen, The complex can be measured by measuring an immune complex of the obtained complex, the reagent antibody, and the bindable substance, and thereby cancer such as primary hepatocellular carcinoma can be determined.
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